Cd, a naturally occurring endocrine toxin found in tobacco leaves, originates in the environment and enters the body through inhalation, targeting the lungs and kidneys. A study published by Larsen-Carey et al. revealed that cadmium mediates the persistence of classically activated lung macrophages to exacerbate lung injury. The research discovered a novel role for PPAR γ as an effective regulator for the alternative activation of macrophages in response to Cd and Cd-induced lung injury.

Due to the ubiquity of environmental micro- and nanoplastics (MNPs), inhalation and ingestion by humans is very likely, but human health effects remain largely unknown. The NLRP3 inflammasome is a key player of the innate immune system and is involved in responses towards foreign particulate matter and the development of chronic intestinal and respiratory inflammatory diseases. We established NLRP3-proficient and -deficient THP-1 cells as an alternative in vitro screening tool to assess the potential of MNPs to activate the NLRP3 inflammasome. By investigating cytokine release (IL-1β and IL-8) and cytotoxicity after treatment with engineered nanomaterials, this in vitro approach was compared to earlier published ex vivo murine bone marrow-derived macrophages and in vivo data. This approach showed a strong correlation with previously published data, verifying that THP-1 cells are a suitable model to investigate NLRP3 inflammasome activation. We then investigated the proinflammatory potential of eight MNPs of different size, shape, and chemical composition. Only amine-modified polystyrene (PS-NH2) acted as a direct NLRP3 activator. However, polyethylene terephthalate (PET), polyacrylonitrile (PAN), and nylon (PA6) induced a significant increase in IL-8 release in NLRP3-/- cells. Our results suggest that most MNPs are not direct activators of the NLRP3 inflammasome, but specific MNP types might still possess pro-inflammatory potential via other pathways.

Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.

The IFITM restriction factors play a role in cancer cell progression through undefined mechanisms. We investigate new protein-protein interactions for IFITM1/3 in the context of cancer that would shed some light on how IFITM1/3 attenuate the expression of targeted proteins such as HLA-B. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the SRSF1 splicing factor and transporter of mRNA to the ribosome. Using in situ proximity ligation assays, we confirmed a predominant cytosolic protein-protein association for SRSF1 and IFITM1/3. Accordingly, IFITM1/3 interacted with HLA-B mRNA in response to IFNγ stimulation using RNA-protein proximity ligation assays. In addition, RT-qPCR assays in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that HLA-B gene expression at the mRNA level does not account for lowered HLA-B protein synthesis in response to IFNγ. Complementary, shotgun RNA sequencing did not show major transcript differences between IFITM1/IFITM3 null cells and wt-SiHa cells. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to wild type. It was partially reverted by IFITM1/3 complementation. Our data link IFITM1/3 proteins to HLA-B mRNA and SRSF1 and, all together, our results begin to elucidate how IFITM1/3 catalyze the synthesis of target proteins. IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. Our study has identified new proteins associated with IFITMs which support their role in mediating protein expression; a pivotal function that is highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 affect the expression of targeted proteins; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs in mediating protein abundance is relevant, as it has the potential for regulating the expression of viral and oncogenic proteins.

Piperidine pharmacophore-containing compounds have demonstrated therapeutic efficacy against a range of diseases and are now being investigated in cancer. A series of 3-chloro-3-methyl-2,6-diarylpiperidin-4-ones, compounds (I-V) were designed and synthesized for their evaluation as a potential anti-cancer agent. Compounds II and IV reduced the growth of numerous hematological cancer cell lines while simultaneously increasing the mRNA expression of apoptosis-promoting genes, p53 and Bax. Molecular docking analyses confirmed that compounds can bind to 6FS1, 6FSO (myeloma), 6TJU (leukemia), 5N21, and 1OLL (NKTL). Computational ADMET research confirmed the essential physicochemical, pharmacokinetic, and drug-like characteristics of compounds (I-V). The results revealed that these compounds interact efficiently with active site residues and that compounds (II) and (V) can be further evaluated as potential therapeutic candidates.

The synthesis, degradation, and reconstruction of the cell membrane as a metabolic pathway of phospholipids is a constant and dynamic process. Fatty acids as bioactive lipid components of plasma and erythrocyte phospholipids as structural lipids have biological roles in the integrity of cell membranes. Fatty acids, depending on the chain length, the degree of saturation, and the synthesis pathways, can alleviate inflammation and oxidative stress caused by excessive exercise. Considering that changing food intake or diet can influence fatty acid phospholipid metabolism, our study aimed to determine the potential benefits of fish-based diets in working (police) dogs undergoing intensive training concerning bioactive lipids such as fatty acids, phospholipids of plasma, and erythrocytes. Fatty acid esters' composition of plasma and erythrocyte phospholipids as a bioactive lipids, in addition to markers of oxidative stress and metabolic parameters, were analysed by GC chromatography. The food was well tolerated by all dogs, and the compliance to the diet was high throughout the study. After the treatment with fish-based food, blood glucose, total, and LDL cholesterol levels were significantly reduced, indicating positive biochemical profiles of dogs. Correlations of fatty acid phospholipid compositions between plasma and erythrocytes have shown that both plasma and erythrocytes could represent markers of omega-3 eicosapentaenoic and docosahexaenoic acid intake levels in dogs. Morover, fish-based food supplementation caused a significant reduction in lipid peroxidation markers. The enrichment of dogs' diets with marine fish could improve oxidative status and improve roles and status of bioactive lipids, such as membrane phospholipids and fatty acids, as its components in plasma and erythrocytes in police dogs exposed to intensive exercise.

Prolonged elevated oxidative stress (OS) possesses negative effect on cell structure and functioning, and is associated with the development of numerous disorders. Naturally occurred anti-oxidant compounds reduce the oxidative stress in living organisms. In this review, antioxidant properties of β-carotene, tocopherols and ascorbic acid are presented based on in vitro, in vivo and populational studies. Firstly, environmental factors contributing to the OS occurrence and intracellular sources of Reactive Oxygen Species (ROS) generation, as well as ROS-mediated cellular structure degradation, are introduced. Secondly, enzymatic and non-enzymatic mechanism of anti-oxidant defence against OS development, is presented. Furthermore, ROS-preventing mechanisms and effectiveness of β-carotene, tocopherols and ascorbic acid as anti-oxidants are summarized, based on studies where different ROS-generating (oxidizing) agents are used. Oxidative stress biomarkers, as indicators on OS level and prevention by anti-oxidant supplementation, are presented with a focus on the methods (spectrophotometric, fluorometric, chromatographic, immuno-enzymatic) of their detection. Finally, the application of Raman spectroscopy and imaging as a tool for monitoring the effect of anti-oxidant (β-carotene, ascorbic acid) on cell structure and metabolism, is proposed. Literature data gathered suggest that β-carotene, tocopherols and ascorbic acid possess potential to mitigate oxidative stress in various biological systems. Moreover, Raman spectroscopy and imaging can be a valuable technique to study the effect of oxidative stress and anti-oxidant molecules in cell studies.

We longitudinally followed a single-center cohort of anti-phospholipid (aPL) positive healthy subjects to evaluate the evolution to systemic autoimmune diseases (sAD) and to describe clinical and serological associated features. Since 2010, we have consecutively screened healthy subjects who were positive, in at least two consecutive determinations, for one or more aPL [anti-Cardiolipin (aCL) IgM/IgG, anti-Beta2Glycoprotein I (aB2GPI) IgM/IgG, Lupus Anticoagulant (LA)]. All subjects were evaluated every six months, or in accordance with the patient's clinical course, in order to record the development of clinical and laboratory features suggestive for sAD. Ninety-five subjects [M/F 20/75, median age at first determination 46 years, Interquartile Range (IQR) 19] were enrolled. Thirty-three subjects (34.7%) were positive for only one aPL [15 (15.8%) for aCL, 15 (15.8%) for LA, and 5 (5.3%) for aB2GPI]; 37 (38.9%) had double positivity [32 (33.6%) for aCL and aB2GPI; 5 (5.3%) for aCL and LA], 23 (24.2%) had triple positivity. We prospectively followed up our cohort for a median period of 72 months (IQR 84). During a total follow-up of 7692 person-months, we found an absolute risk for sAD development equal to 1.8%. Specifically, 14 (14.7%) patients developed a sAD: in four patients (4.2%), after developing a thrombotic event, an antiphospholipid syndrome was diagnosed, 7 (7.4%) patients developed an Undifferentiated Connective Tissue Disease after a median period of 76 months (IQR 75.5), and lastly, three (3.1%) patients could be classified as affected by Systemic Lupus Erythematosus according to the ACR/EULAR 2019 criteria. The presence of triple positivity status resulted in being significantly associated with the progression to sAD (p-value = 0.03). In conclusion, we observed the development of sAD in almost 15% of aPL positive subjects. Triple positivity was significantly associated with this progression, suggesting a possible role as biomarker for this condition. Thus, our results could suggest the need for periodic follow-up for such patients to assess early diagnosis and treatment.

In recent years, multifunctional hydrogels have received a great deal of attention because they are biocompatible and can mimic the extracellular matrix. Herein, we prepared hydrogels of biocompatible cross-linked networks with photothermal properties. In this study, a chitosan/polyaniline/laponite (COL) hydrogel with photothermal conversion capability was designed. Polyaniline was firstly grafted onto chitosan and its solution was mixed with oxidized dextran, which was then cross-linked into a hydrogel via a Schiff base reaction. Furthermore, an aluminosilicate clay material, laponite (LAP), was incorporated into the hydrogel. The swelling ratio of the COL hydrogel in various solutions was greater than 580%, and it showed good degradation ability (the mass-loss ratio was over 45% after 28 days). This composite hydrogel was demonstrated to have good photothermal conversion properties and biocompatibility at both the cell (cell viability was over 97%) and animal levels. The COL hydrogel showed a photothermal conversion efficiency of 23.7% under the irradiation of a near-infrared laser. Coupled with the osteogenic differentiation-inducing potential of LAP, the COL hydrogel has the potential to kill tumors via hyperthermia or serve as scaffolds for bone tissue regeneration.

Methylation is a crucially important ubiquitous biochemical process, which covalently adds methyl groups to a variety of molecular targets. It is the key regulatory process that determines the acquisition of imprinting and epigenetic marks during gametogenesis. Methylation processes are dependent upon two metabolic cycles, the folates and the one-carbon cycles. The activity of these two cycles is compromised by single nucleotide polymorphisms (SNPs) in the gene encoding the Methylenetetrahydrofolate reductase (MTHFR) enzyme. These SNPs affect spermatogenesis and oocyte maturation, creating cytologic/chromosomal anomalies. The two main MTHFR SNP variants C677T (c.6777C>T) and A1298C (c.1298A>C) together with serum homocysteine levels were tested in men with >3 years' duration of infertility who had failed several ART attempts with the same partner. These patients are often classified as having "idiopathic infertility". We observed that the genetic status with highest prevalence in this group is the heterozygous C677T, followed by the combined heterozygous C677T/A1298C, and then A1298C; these three variants represent 65% of our population. Only 13.1% of the patients tested are wild type (WT), C677C/A1298A). The homozygous 677TT and the combined heterozygote 677CT/1298AC groups have the highest percentage of patients with an elevated circulating homocysteine level of >15 µMolar (57.8% and 18.8%, respectively, which is highly significant for both). Elevated homocysteine is known to be detrimental to spermatogenesis, and the population with this parameter is not marginal. In conclusion, determination of these two SNPs and serum homocysteine should not be overlooked for patients with severe infertility of long duration, including those with repeated miscarriages. Patients must also be informed about pleiotropic medical implications relevant to their own health, as well as to the health of future children.

High concentrations of aldehydes may result in poor-quality agricultural distillate. We investigate the influence of the method of mash preparation, the initial pH of the mashes, and different yeast strains on the fermentation efficiency and concentration of aldehydes from C2 (acetaldehyde) to C7 (enanthaldehyde) in rye mashes. The tested factors were revealed to have a differentiated influence on both the process efficiency and the concentrations of aldehydes, especially in the case of the dominant acetaldehyde. Mashes obtained from steamed rye grain showed significantly higher fermentation efficiencies than those prepared by the pressureless method. Increasing the pH of the sweet mashes from 4.5 to 6.0 resulted in significantly higher concentrations of acetaldehyde, especially in the case of steamed rye grain. Moreover, an increase in the concentrations of other aldehydes, i.e., from C3 (propionaldehyde) to C5 (valer- and isovaleraldehyde) was observed. A high fermentation efficiency and the lowest acetaldehyde concentrations were obtained from steamed rye mashes with an initial pH of 4.5, fermented using the yeast strains DistilaMax GW and DistilaMax HT. DistilaMax HT yeast also provided a relatively low concentration of acetaldehyde in mashes with an initial pH in the range of 4.5-5.5 prepared by the energy-saving pressureless method.

Despite the very large number of phytocannabinoids isolated from Cannabis (Cannabis sativa L.), bioactivity studies have long remained focused on the so called "Big Four" [Δ9-THC (1), CBD (2), CBG (3) and CBC (4)] because of their earlier characterization and relatively easy availability via isolation and/or synthesis. Bioactivity information on the chemical space associated with the remaining part of the cannabinome, a set of ca 150 compounds traditionally referred to as "minor phytocannabinoids", is scarce and patchy, yet promising in terms of pharmacological potential. According to their advancement stage, we sorted the bioactivity data available on these compounds, better referred to as the "dark cannabinome", into categories: discovery (in vitro phenotypical and biochemical assays), preclinical (animal models), and clinical. Strategies to overcome the availability issues associated with minor phytocannabinoids are discussed, as well as the still unmet challenges facing their development as mainstream drugs.

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.

The Warburg effect, i.e., the utilization of glycolysis under aerobic conditions, is recognized as a survival advantage of cancer cells. However, how the glycolytic activity is affected during drug resistance acquisition has not been explored at single-cell resolution. Because the relative ratio of the splicing isoform of pyruvate kinase M (PKM), PKM2/PKM1, can be used to estimate glycolytic activity, we utilized a single-molecule fluorescence in situ hybridization (SM-FISH) method to simultaneously quantify the mRNA levels of PKM1 and PKM2. Treatment of HCT116 cells with gefitinib (GE) resulted in two distinct populations of cells. However, as cells developed GE resistance, the GE-sensitive population with reduced PKM2 expression disappeared, and GE-resistant cells (Res) demonstrated enhanced PKM1 expression and a tightly regulated PKM2/PKM1 ratio. Our data suggest that maintaining an appropriate PKM2 level is important for cell survival upon GE treatment, whereas increased PKM1 expression becomes crucial in GE Res. This approach demonstrates the importance of single-cell-based analysis for our understanding of cancer cell metabolic responses to drugs, which could aid in the design of treatment strategies for drug-resistant cancers.

Colon adenocarcinoma is one of the tumors with the highest mortality rate, and tumorigenesis or development of colon adenocarcinoma is the major reason leading to patient death. However, the molecular mechanism and biomarker to predict tumor progression are currently unclear. With the goal of understanding the molecular mechanism and tumor progression, we utilized the TCGA database to identify differentially expressed genes. After identifying the differentially expressed genes among colon adenocarcinoma tissues with different expression levels of LGR4 and normal tissue, protein-protein interaction, gene ontology, pathway enrichment, gene set enrichment analysis, and immune cell infiltration analysis were conducted. Here, the top 10 hub genes, i.e., ALB, F2, APOA2, CYP1A1, SPRR2B, APOA1, APOB, CYP3A4, SST, and GCG, were identified, and relative correlation analysis was conducted. Kaplan-Meier analysis revealed that higher expression of LGR4 correlates with overall survival of colon adenocarcinoma patients, although expression levels of LGR4 in normal tissues are higher than in tumor tissues. Further functional analysis demonstrated that higher expression of LGR4 in colon adenocarcinoma may be linked to up-regulate metabolism-related pathways, for example, the cholesterol biosynthesis pathway. These results were confirmed by gene set enrichment analysis. Immune cell infiltration analysis clearly showed that the infiltration percentage of T cells was significantly higher than other immune cells, and TIMER analysis revealed a positive correlation between T-cell infiltration and LGR4 expression. Finally, COAD cancer cells, Caco-2, were employed to be incubated with squalene and 25-hydroxycholesterol-3-sulfate, and relative experimental results confirmed that the cholesterol biosynthesis pathway involved in modulating the proliferation of COAD tumorigenesis. Our investigation revealed that LGR4 can be an emerging diagnostic and prognostic biomarker for colon adenocarcinoma by affecting metabolism-related pathways.

Serum ferritin (SF) is an iron-rich protein tightly connected with iron homeostasis, and the variations are frequently observed in diseased states, including iron-deficiency anemia, inflammation, liver disease, and tumors, which renders SF level an indicator of potential malignancies in clinical practice. Nanobodies (Nbs) have been widely explored and developed into theranostic reagents. Surprisingly, no reports stated the identification of anti-SF Nbs, nor the potential of such Nbs as a diagnostic tool. In this study, we generated SF-specific Nbs and provided novel clinical diagnostic approaches to develop an immunoassay. An immune library was constructed after immunizing an alpaca with SF, and five Nbs specifically targeting human SF were retrieved. The obtained Nbs exhibited robust properties including high stability, affinity, and specificity. Then, an ELISA-based test using a heterologous Nb-pair was developed. The calibration curve demonstrated a linear range of SF between 9.0 to 1100 ng/mL, and a limit of detection (LOD) of 1.01 ng/mL. The detecting recovery and coefficient variation (CV) were determined by spiking different concentrations of SF into the serum sample, to verify the successful application of our selected Nbs for SF monitoring. In general, this study generated SF-specific Nbs and demonstrated their potential as diagnostic immunoassay tools.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance, mitochondrial dysfunction, inflammation, and oxidative stress. As a group of NAD+-dependent III deacetylases, the sirtuin (SIRT1-7) family plays a very important role in regulating mitochondrial biogenesis and participates in the progress of NAFLD. SIRT family members are distributed in the nucleus, cytoplasm, and mitochondria; regulate hepatic fatty acid oxidation metabolism through different metabolic pathways and mechanisms; and participate in the regulation of mitochondrial energy metabolism. SIRT1 may improve NAFLD by regulating ROS, PGC-1α, SREBP-1c, FoxO1/3, STAT3, and AMPK to restore mitochondrial function and reduce steatosis of the liver. Other SIRT family members also play a role in regulating mitochondrial biogenesis, fatty acid oxidative metabolism, inflammation, and insulin resistance. Therefore, this paper comprehensively introduces the role of SIRT family in regulating mitochondrial biogenesis in the liver in NAFLD, aiming to further explain the importance of SIRT family in regulating mitochondrial function in the occurrence and development of NAFLD, and to provide ideas for the research and development of targeted drugs. Relatively speaking, the role of some SIRT family members in NAFLD is still insufficiently clear, and further research is needed.

The highly conserved molecular chaperone heat shock protein 90 (Hsp90) is well-known for maintaining metastable proteins and mediating various aspects of intracellular protein dynamics. Intriguingly, high-throughput interactome studies suggest that Hsp90 is associated with a variety of other pathways. Here, we will highlight the potential impact of Hsp90 in protein transport. Currently, a limited number of studies have defined a few mechanistic contributions of Hsp90 to protein transport, yet the relevance of hundreds of additional connections between Hsp90 and factors known to aide this process remains unresolved. These interactors broadly support transport pathways including endocytic and exocytic vesicular transport, the transfer of polypeptides across membranes, or unconventional protein secretion. In resolving how Hsp90 contributes to the protein transport process, new therapeutic targets will likely be obtained for the treatment of numerous human health issues, including bacterial infection, cancer metastasis, and neurodegeneration.

Cisplatin is an FDA approved anti-cancer drug that is widely used for the treatment of a variety of solid tumors. However, the severe adverse effects of cisplatin, particularly kidney toxicity, restrict its clinical and medication applications. The major mechanisms of cisplatin-induced renal toxicity involve oxidative stress, inflammation, and renal fibrosis, which are covered in this short review. In particular, we review the underlying mechanisms of cisplatin kidney injury in the context of NAD+-dependent redox enzymes including mitochondrial complex I, NAD kinase, CD38, sirtuins, poly-ADP ribosylase polymerase, and nicotinamide nucleotide transhydrogenase (NNT) and their potential contributing roles in the amelioration of cisplatin-induced kidney injury conferred by natural products derived from plants. We also cover general procedures used to create animal models of cisplatin-induced kidney injury involving mice and rats. We highlight the fact that more studies will be needed to dissect the role of each NAD+-dependent redox enzyme and its involvement in modulating cisplatin-induced kidney injury, in conjunction with intensive research in NAD+ redox biology and the protective effects of natural products against cisplatin-induced kidney injury.

The consequences of aging and disease conditions in tissues involve reactive oxygen species (ROS) and related molecular alterations of different cellular compartments. We compared a murine model of immunodeficient (SCID) xenografted young (4 weeks old) and old (17 weeks old) mice with corresponding controls without tumor implantation and carried out a compositional evaluation of brain tissue for changes in parallel DNA and lipids compartments. DNA damage was measured by four purine 5',8-cyclo-2'-deoxynucleosides, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA). In brain lipids, the twelve most representative fatty acid levels, which were mostly obtained from the transformation of glycerophospholipids, were followed up during the aging and disease progressions. The progressive DNA damage due to age and tumoral conditions was confirmed by raised levels of 5'S-cdG and 5'S-cdA. In the brain, the remodeling involved a diminution of palmitic acid accompanied by an increase in arachidonic acid, along both age and tumor progressions, causing increases in the unsaturation index, the peroxidation index, and total TFA as indicators of increased oxidative and free radical reactivity. Our results contribute to the ongoing debate on the central role of DNA and genome instability in the aging process, and on the need for a holistic vision, which implies choosing the best biomarkers for such monitoring. Furthermore, our data highlight brain tissue for its lipid remodeling response and inflammatory signaling, which seem to prevail over the effects of DNA damage.

Atomic-resolution structural studies of membrane-associated proteins and peptides in a membrane environment are important to fully understand their biological function and the roles played by them in the pathology of many diseases. However, the complexity of the cell membrane has severely limited the application of commonly used biophysical and biochemical techniques. Recent advancements in NMR spectroscopy and cryoEM approaches and the development of novel membrane mimetics have overcome some of the major challenges in this area. For example, the development of a variety of lipid-nanodiscs has enabled stable reconstitution and structural and functional studies of membrane proteins. In particular, the ability of synthetic amphipathic polymers to isolate membrane proteins directly from the cell membrane, along with the associated membrane components such as lipids, without the use of a detergent, has opened new avenues to study the structure and function of membrane proteins using a variety of biophysical and biological approaches. This review article is focused on covering the various polymers and approaches developed and their applications for the functional reconstitution and structural investigation of membrane proteins. The unique advantages and limitations of the use of synthetic polymers are also discussed.

Intervertebral disc degeneration (IVDD) is a common musculoskeletal degenerative disease worldwide, of which the main clinical manifestation is low back pain (LBP); approximately, 80% of people suffer from it in their lifetime. Currently, the pathogenesis of IVDD is unclear, and modern treatments can only alleviate its symptoms but cannot inhibit or reverse its progression. However, in recent years, targeted therapy has led to new therapeutic strategies. Cysteine-containing aspartate proteolytic enzymes (caspases) are a family of proteases present in the cytoplasm. They are evolutionarily conserved and are involved in cell growth, differentiation, and apoptotic death of eukaryotic cells. In recent years, it has been confirmed to be involved in the pathogenesis of various diseases, mainly by regulating cell apoptosis and inflammatory response. With continuous research on the pathogenesis and pathological process of IVDD, an increasing number of studies have shown that caspases are closely related to the IVDD process, especially in the intervertebral disc (IVD) cell apoptosis and inflammatory response. Therefore, herein we study the role of caspases in IVDD with respect to the structure of caspases and the related signaling pathways involved. This would help explore the strategy of regulating the activity of the caspases involved and develop caspase inhibitors to prevent and treat IVDD. The aim of this review was to identify the caspases involved in IVDD which could be potential targets for the treatment of IVDD.

Virus discovery has been fueled by new technologies ever since the first viruses were discovered at the end of the 19th century. Starting with mechanical devices that provided evidence for virus presence in sick hosts, virus discovery gradually transitioned into a sequence-based scientific discipline, which, nowadays, can characterize virus identity and explore viral diversity at an unprecedented resolution and depth. Sequencing technologies are now being used routinely and at ever-increasing scales, producing an avalanche of novel viral sequences found in a multitude of organisms and environments. In this perspective article, we argue that virus discovery has started to undergo another transformation prompted by the emergence of new approaches that are sequence data-centered and primarily computational, setting them apart from previous technology-driven innovations. The data-driven virus discovery approach is largely uncoupled from the collection and processing of biological samples, and exploits the availability of massive amounts of publicly and freely accessible data from sequencing archives. We discuss open challenges to be solved in order to unlock the full potential of data-driven virus discovery, and we highlight the benefits it can bring to classical (mostly molecular) virology and molecular biology in general.

Hypoxia plays a crucial role in acute and chronic renal injury, which is attributable to renal tubular and glomerular cell damage. Some studies provide evidence that hypoxia-dependent upregulation of the mitochondrial enzyme arginase type-II (Arg-II) in tubular cells promotes renal tubular injury. It is, however, not known whether Arg-II is also expressed in glomerular cells, particularly podocytes under hypoxic conditions, contributing to hypoxia-induced podocyte injury. The effects of hypoxia on human podocyte cells (AB8/13) in cultures and on isolated kidneys from wild-type (wt) and arg-ii gene-deficient (arg-ii-/-) mice ex vivo, as well as on mice of the two genotypes in vivo, were investigated, respectively. We found that the Arg-II levels were enhanced in cultured podocytes in a time-dependent manner over 48 h, which was dependent on the stabilization of hypoxia-inducible factor 1α (HIF1α). Moreover, a hypoxia-induced derangement of cellular actin cytoskeletal fibers, a decrease in podocin, and an increase in mitochondrial ROS (mtROS) generation-as measured by MitoSOX-were inhibited by adenoviral-mediated arg-ii gene silencing. These effects of hypoxia on podocyte injury were mimicked by the HIFα stabilizing drug DMOG, which inhibits prolyl hydroxylases (PHD), the enzymes involved in HIFα degradation. The silencing of arg-ii prevented the detrimental effects of DMOG on podocytes. Furthermore, the inhibition of mtROS generation by rotenone-the inhibitor of respiration chain complex-I-recapitulated the protective effects of arg-ii silencing on podocytes under hypoxic conditions. Moreover, the ex vivo experiments with isolated kidney tissues and the in vivo experiments with mice exposed to hypoxic conditions showed increased Arg-II levels in podocytes and decreased podocyte markers regarding synaptopodin in wt mice but not in arg-ii-/- mice. While age-associated albuminuria was reduced in the arg-ii-/- mice, the hypoxia-induced increase in albuminuria was, however, not significantly affected in the arg-ii-/-. Our study demonstrates that Arg-II in podocytes promotes cell injury. Arg-ii ablation seems insufficient to protect mice in vivo against a hypoxia-induced increase in albuminuria, but it does reduce albuminuria in aging.

Prothymosin α (PTMA) is a phylogenetically conserved polypeptide in male gonads of Vertebrates. In Mammals, it is a ubiquitous protein, and, possessing a random-coil structure, it interacts with many other partners, in both cytoplasmic and nuclear compartments. PTMA has been widely studied during cell progression in different types of cancer because of its anti-apoptotic and proliferative properties. Here, we provided the first evidence of PTMA expression and localization in human testis and in two testicular cancers (TC): classic seminoma (CS) and Leydig cell tumor (LCT). Data showed that its protein level, together with that of proliferating cell nuclear antigen (PCNA), a cell cycle progression marker, increased in both CS and LCT samples, as compared to non-pathological (NP) tissue. Moreover, in the two-cancer tissue, a decreased apoptotic rate and an increased autophagic flux was also evidenced. Results confirmed the anti-apoptotic action of PTMA, also suggesting that it can act as a switcher from apoptosis to autophagy, to favor the survival of testicular cancer cells when they develop in adverse environments. Finally, the combined data, even if they need to be further validated, add new insight into the role of PTMA in human normal and pathological testicular tissue.

Piezo2 transmembrane excitatory mechanosensitive ion channels were identified as the principal mechanotransduction channels for proprioception. Recently, it was postulated that Piezo2 channels could be acutely microdamaged on an autologous basis at proprioceptive Type Ia terminals in a cognitive demand-induced acute stress response time window when unaccustomed or strenuous eccentric contractions are executed. One consequence of this proposed transient Piezo2 microinjury could be a VGLUT1/Ia synaptic disconnection on motoneurons, as we can learn from platinum-analogue chemotherapy. A secondary, harsher injury phase with the involvement of polymodal Aδ and nociceptive C-fibers could follow the primary impairment of proprioception of delayed onset muscle soreness. Repetitive reinjury of these channels in the form of repeated bout effects is proposed to be the tertiary injury phase. Notably, the use of proprioception is associated with motor learning and memory. The impairment of the monosynaptic static phase firing sensory encoding of the affected stretch reflex could be the immediate consequence of the proposed Piezo2 microdamage leading to impaired proprioception, exaggerated contractions and reduced range of motion. These transient Piezo2 channelopathies in the primary afferent terminals could constitute the critical gateway to the pathophysiology of delayed onset muscle soreness. Correspondingly, fatiguing eccentric contraction-based pathological hyperexcitation of the Type Ia afferents induces reactive oxygen species production-associated neuroinflammation and neuronal activation in the spinal cord of delayed onset muscle soreness.

2-Hydroxypropyl-beta-cyclodextrin (2HPβCD) is able to bind and solubilize unesterified cholesterol and may therefore be able to reverse the deposition of cholesterol in macrophages within the aortic vessel wall, a hallmark of atherosclerotic cardiovascular disease. However, conflicting results regarding the potential of 2HPβCD to induce regression of established atherosclerotic lesions have been described. In the current study, we therefore also investigated the ability of 2HPβCD to stimulate cholesterol removal from macrophage foam cells in vitro and induce the regression of established atherosclerotic lesions in apolipoprotein E knockout (APOE KO) mice. In vitro studies using murine thioglycollate-elicited peritoneal macrophages verified that 2HPβCD is able to induce cholesterol efflux from macrophages in an ATP-binding cassette transporter-independent manner. Switching Western-type-diet-fed APOE KO mice with established atherosclerotic lesions back to a chow diet was associated with a reduction in the hypercholesterolemia extent and an increase in the absolute lesion size and plaque collagen-to-macrophage ratio. Importantly, parallel subcutaneous administration of 2HPβCD was not able to prevent the diet-switch-associated lesion growth or induce atherosclerosis regression. Although in our hands, 2HPβCD does effectively stimulate cellular cholesterol efflux from macrophages, we do not consider it worthwhile to further pursue 2HPβCD as therapeutic moiety in the atherosclerosis regression context.

De novo sterol synthesis is a critical homeostatic mechanism in the brain that begins during early embryonic development and continues throughout life. Multiple medications have sterol-biosynthesis-inhibiting side effects, with potentially detrimental effects on brain health. Using LC-MS/MS, we investigated the effects of six commonly used beta-blockers on brain sterol biosynthesis in vitro using cell lines. Two beta-blockers, metoprolol (MTP) and nebivolol, showed extreme elevations of the highly oxidizable cholesterol precursor 7-dehydrocholesterol (7-DHC) in vitro across multiple cell lines. We followed up on the MTP findings using a maternal exposure model in mice. We found that 7-DHC was significantly elevated in all maternal brain regions analyzed as well as in the heart, liver and brain of the maternally exposed offspring. Since DHCR7-inhibiting/7-DHC elevating compounds can be considered teratogens, these findings suggest that MTP utilization during pregnancy might be detrimental for the development of offspring, and alternative beta-blockers should be considered.

Plants of the species Fridericia chica (Bonpl.) L. G. Lohmann (Bignoniaceae), which are widely distributed in Brazil and named crajiru in the state of Amazonas, are known in folk medicine as a traditional medicine in the form of a tea for the treatment of intestinal colic, diarrhea, and anemia, among other diseases. The chemical analysis of extracts of the leaves has identified phenolic compounds, a class of secondary metabolites that provide defense for plants and benefits to the health of humans. Several studies have shown the therapeutic efficacy of F. chica extracts, with antitumor, antiviral, wound healing, anti-inflammatory, and antioxidant activities being among the therapeutic applications already proven. The healing action of F. chica leaf extract has been demonstrated in several experimental models, and shows the ability to favor the proliferation of fibroblasts, which is essential for tissue repair. The anti-inflammatory activity of F. chica has been clearly demonstrated by several authors, who suggest that it is related to the presence of 3-deoxyanthocyanidins, which is capable of inhibiting pro-inflammatory pathways such as the kappa B (NF-kB) nuclear transcription factor pathway. Another important effect attributed to this species is the antioxidant effect, attributed to phenolic compounds interrupting chain reactions caused by free radicals and donating hydrogen atoms or electrons. In conclusion, the species Fridericia chica has great therapeutic potential, which is detailed in this paper with the objective of encouraging new research and promoting the sum of efforts for the inclusion of herbal medicines in health systems around the world.

The SARS-CoV-2 pandemic has disrupted global health systems and brought the entire globe to its knees. Although born as a disease of the respiratory system, COVID-19 can affect different parts of the body, including the skin. Reports of ongoing skin manifestations of COVID-19 have gradually multiplied, pushing researchers to investigate the etiopathogenic mechanisms underlying these phenomena in more depth. In an attempt to investigate the possible association between SARS-CoV-2, ACE2, TMPRSS2 and skin manifestations, we performed immunohistochemical investigations of the ACE2 receptor and TMPRSS2 in nine skin samples from SARS-CoV-2-positive patients compared to a cohort of healthy controls. Furthermore, after consulting public databases regarding ACE2 mRNA expression in various cell populations resident in the skin, we conducted a literature review aimed at outlining the current state of this topic. We did not find statistically different immuno-expression of ACE2 and TMPRSS2 between the group of SARS-CoV-2-positive patients (nine skin biopsies) and the control group. Regarding ACE2, major immunolabeling was present in the epidermal keratinocytes and, rarely, in the fibroblasts and in the adenomeres of the eccrine sweat glands. Regarding the immune expression of TMPRSS2, we found no significant differences between the two groups, with a weak immune staining only in some skin cytotypes. From the review of the literature, we isolated 35 relevant articles according to the inclusion criteria adopted. ACE2 appears to be a target of SARS-CoV-2, although, other receptor molecules may potentially be implicated, such as TMPRSS2. Future studies with large cases and different molecular investigative methods are needed to further elucidate the mechanisms underlying the skin manifestations of SARS-CoV-2.

Agonistic antibodies targeting co-stimulating receptor OX40 on T cells are considered as important as (or complementary to) the immune checkpoint blockers in cancer treatment. However, none of these agonistic antibodies have reached the late stage of clinical development partially due to the lack of intrinsic potency with the correlation between binding epitope and activity of the antibody not well understood. Here, we identified a novel anti-OX40 agonistic antibody DF004, which stimulated the proliferation of human CD4+ T cells in vitro and inhibited tumor growth in a mouse model. Our crystallography structural studies showed that DF004 binds to the CRD2 region of OX40 while RG7888, an OX40 agonist antibody developed by Roche, binds to CRD3 of OX40 to the diametrically opposite position of DF004. This suggests that the agonistic activities of the antibodies are not necessarily epitope dependent. As their agonistic activities critically depend on clustering or cross-linking, our structural modeling indicates that the agonistic activity requires the optimal positioning of three Fc receptor/antibody/OX40 complexes on the cell membrane to facilitate the formation of one intracellular hexameric TRAF complex for downstream signal transduction, which is relatively inefficient. This may explain the lack of sufficient potency of these OX40 antibodies in a therapeutic setting and sheds light on the development of cross-linking-independent agonistic antibodies.

Cisplatin (cis-diamminedichloroplatinum (II), DDP) is an antineoplastic agent widely used in the treatment of solid tumors because of its extensive cytotoxic activity. However, the main limiting side effect of DDP use is nephrotoxicity, a rapid deterioration in kidney function due to toxic chemicals. Several studies have shown that epigenetic processes are involved in DDP-induced nephrotoxicity. Noncoding RNAs (ncRNAs), a class of epigenetic processes, are molecules that regulate gene expression under physiological and pathological conditions. MicroRNAs (miRNAs) are the most characterized class of ncRNAs and are engaged in many cellular processes. In this review, we describe how different miRNAs regulate some pathways leading to cell death by apoptosis, specifically the intrinsic apoptosis pathway. Accordingly, many classes of natural products have been tested for their ability to prevent DDP-induced apoptosis. The study of epigenetic regulation for underlying cell death is still being studied, which will allow new strategies for the diagnosis and therapy of this unwanted disease, which is presented as a side effect of antineoplastic treatment.

Accumulating data suggests that catestatin, an eclectic neuroendocrine peptide, is involved in the pathophysiology of primary hypertension (PH). Nevertheless, clinical studies concerning its role in PH are still scarce. Therefore, in the present study, we aimed to explore an association between serum catestatin levels, ambulatory blood pressure (BP) and arterial stiffness in patients with PH and healthy controls. In this single-center study, 72 patients aged 40-70 diagnosed with PH, and 72 healthy controls were included. In patients with PH, serum catestatin concentrations were significantly higher in comparison to the healthy controls (29.70 (19.33-49.48) ng/mL vs. 5.83 (4.21-8.29) ng/mL, p < 0.001). Untreated patients had significantly higher serum catestatin than patients treated with antihypertensive drugs (41.61 (22.85-63.83) ng/mL vs. 24.77 (16.41-40.21) ng/mL, p = 0.005). Multiple linear regression analysis showed that serum catestatin levels retained a significant association with mean arterial pressure (β ± standard error, 0.8123 ± 0.3037, p < 0.009) after model adjustments for age, sex and body mass index. Finally, catestatin levels positively correlated with pulse wave velocity (r = 0.496, p < 0.001) and central augmentation index (r = 0.441, p < 0.001), but not with peripheral resistance. In summary, increased serum catestatin concentration in PH, predominantly in the untreated subgroup, and its association with ambulatory BP and arterial stiffness address the role of this peptide in PH.

Sex differences are increasingly being explored and reported in oncology, and glioma is no exception. As potentially meaningful sex differences are uncovered, existing gender-derived disparities mirror data generated in retrospective and prospective trials, real-world large-scale data sets, and bench work involving animals and cell lines. The resulting disparities at the data level are wide-ranging, potentially resulting in both adverse outcomes and failure to identify and exploit therapeutic benefits. We set out to analyze the literature on women's data disparities in glioma by exploring the origins of data in this area to understand the representation of women in study samples and omics analyses. Given the current emphasis on inclusive study design and research, we wanted to explore if sex bias continues to exist in present-day data sets and how sex differences in data may impact conclusions derived from large-scale data sets, omics, biospecimen analysis, novel interventions, and standard of care management.

The precise regulation of metabolism and feeding behavior is important for preventing the development of metabolic diseases. Here we examine the effects on Drosophila metabolism of dietary choice. These changes are predicted to be dependent on both the quantity and quality of the chosen diet. Using a geometric framework for both no-choice and two-choice conditions, we found that feeding decisions led to higher glucose and trehalose levels but lower triglycerides pools. The feeding regimens had similar strategies for macronutrient balancing, and both maximized hemolymph glucose and glycogen content under low protein intake. In addition, the flies showed significant differences in the way they regulated trehalose and triglyceride levels in response to carbohydrate and protein consumption between choice and no-choice nutrition. Under choice conditions, trehalose and triglyceride levels were maximized at the lowest protein and carbohydrate consumption. Thus, we suggest that these changes in carbohydrate and lipid metabolism are caused by differences in the macronutrients consumed by flies. Food choice elicits rapid metabolic changes to maintain energy homeostasis. These results contribute to our understanding of how metabolism is regulated by the revealed nutrient variation in response to food decisions.

Natural products (NPs) are a rich source of structurally novel molecules, and the chemical space they encompass is far from being fully explored. Over history, NPs have represented a significant source of bioactive molecules and have served as a source of inspiration for developing many drugs on the market. On the other hand, computer-aided drug design (CADD) has contributed to drug discovery research, mitigating costs and time. In this sense, compound databases represent a fundamental element of CADD. This work reviews the progress toward developing compound databases of natural origin, and it surveys computational methods, emphasizing chemoinformatic approaches to profile natural product databases. Furthermore, it reviews the present state of the art in developing Latin American NP databases and their practical applications to the drug discovery area.

Skin inflammation occurs due to immune dysregulation because of internal disorders, infections, and allergic reactions. The inflammation of the skin is a major sign of chronic autoimmune inflammatory diseases, such as psoriasis, atopic dermatitis (AD), and lupus erythematosus. Although there are many therapies for treating these cutaneous inflammation diseases, their recurrence rates are high due to incomplete resolution. MicroRNA (miRNA) plays a critical role in skin inflammation by regulating the expression of protein-coding genes at the posttranscriptional level during pathogenesis and homeostasis maintenance. Some miRNAs possess anti-inflammatory features, which are beneficial for mitigating the inflammatory response. miRNAs that are reduced in inflammatory skin diseases can be supplied transiently using miRNA mimics and agomir. miRNA-based therapies that can target multiple genes in a given pathway are potential candidates for the treatment of skin inflammation. This review article offers an overview of the function of miRNA in skin inflammation regulation, with a focus on psoriasis, AD, and cutaneous wounds. Some bioactive molecules can target and modulate miRNAs to achieve the objective of inflammation suppression. This review also reports the anti-inflammatory efficacy of these molecules through modulating miRNA expression. The main limitations of miRNA-based therapies are rapid biodegradation and poor skin and cell penetration. Consideration was given to improving these drawbacks using the approaches of cell-penetrating peptides (CPPs), nanocarriers, exosomes, and low-frequency ultrasound. A formulation design for successful miRNA delivery into skin and target cells is also described in this review. The possible use of miRNAs as biomarkers and therapeutic modalities could open a novel opportunity for the diagnosis and treatment of inflammation-associated skin diseases.

1,3-diaryl-2-propanone derivatives are synthetic compounds used as building blocks for the realization not only of antimicrobial drugs but also of new nanomaterials thanks to their ability to self-assemble in solution and interact with nucleopeptides. However, their ability to interact with proteins is a scarcely investigated theme considering the therapeutic importance that 1,3-diaryl-2-propanones could have in the modulation of protein-driven processes. Within this scope, we investigated the protein binding ability of 1,3-bis(1'-uracilyl)-2-propanone, which was previously synthesized in our laboratory utilizing a Dakin-West reaction and herein indicated as U2O, using bovine serum albumin (BSA) as the model protein. Through circular dichroism (CD) and UV spectroscopy, we demonstrated that the compound, but not the similar thymine derivative T2O, was able to alter the secondary structure of the serum albumin leading to significant consequences in terms of BSA structure with respect to the unbound protein (Δβ-turn + Δβ-sheet = +23.6%, Δα = -16.7%) as revealed in our CD binding studies. Moreover, molecular docking studies suggested that U2O is preferentially housed in the domain IIIB of the protein, and its affinity for the albumin is higher than that of the reference ligand HA 14-1 (HDOCK score (top 1-3 poses): -157.11 ± 1.38 (U2O); -129.80 ± 6.92 (HA 14-1); binding energy: -7.6 kcal/mol (U2O); -5.9 kcal/mol (HA 14-1)) and T2O (HDOCK score (top 1-3 poses): -149.93 ± 2.35; binding energy: -7.0 kcal/mol). Overall, the above findings suggest the ability of 1,3-bis(1'-uracilyl)-2-propanone to bind serum albumins and the observed reduction of the α-helix structure with the concomitant increase in the β-structure are consistent with a partial protein destabilization due to the interaction with U2O.

Reducing the health hazards caused by air pollution is a global challenge and is included in the Sustainable Development Goals. Air pollutants, such as PM2.5, induce respiratory and cardiovascular disorders by causing various inflammatory responses via oxidative stress. Catechins and polyphenols, which are components of green tea, have various protective effects, owing to their antioxidant ability. The main catechin in green tea, epigallocatechin gallate (EGCG), is potentially effective against respiratory diseases, such as idiopathic pulmonary fibrosis and asthma, but its effectiveness against air-pollution-dependent lung injury has not yet been investigated. In this study, we examined the effect of EGCG on urban aerosol-induced acute lung injury in mice. Urban aerosol treatment caused increases in inflammatory cell counts, protein levels, and inflammatory cytokine expression in the lungs of ICR mice, but pretreatment with EGCG markedly suppressed these responses. Analyses of oxidative stress revealed that urban aerosol exposure enhanced reactive oxygen species (ROS) production and the formation of ROS-activated neutrophil extracellular traps (NETs) in the lungs of mice. However, ROS production and NETs formation were markedly suppressed by pretreating the mice with EGCG. Gallocatechin gallate (GCG), a heat-epimerized form of EGCG, also markedly suppressed urban aerosol-dependent inflammatory responses and ROS production in vivo and in vitro. These findings suggest that EGCG and GCG prevent acute lung injury caused by urban aerosols through their inhibitory effects on ROS production. Thus, we believe that foods and medications containing EGCG or GCG may be candidates to prevent the onset and progression of acute lung injury caused by air pollutants.

α-synuclein is a core component of Lewy bodies, one of the pathological hallmarks of Parkinson's disease. Aggregated α-synuclein can impair both synaptic functioning and axonal transport. However, understanding the pathological role that α-synuclein plays at a cellular level is complicated as existing findings are multifaceted and dependent on the mutation, the species, and the quantity of the protein that is involved. This systematic review aims to stratify the research findings to develop a more comprehensive understanding of the role of aggregated α-synuclein on synaptic and axonal proteins in Parkinson's disease models. A literature search of the PubMed, Scopus, and Web of Science databases was conducted and a total of 39 studies were included for analysis. The review provides evidence for the dysregulation or redistribution of synaptic and axonal proteins due to α-synuclein toxicity. However, due to the high quantity of variables that were used in the research investigations, it was challenging to ascertain exactly what effect α-synuclein has on the expression of the proteins. A more standardized experimental approach regarding the variables that are employed in future studies is crucial so that existing literature can be consolidated. New research involving aggregated α-synuclein at the synapse and regarding axonal transport could be advantageous in guiding new treatment solutions.

Hyperthyroidism is common and can induce cardiomyopathy, but there is no effective therapeutic strategy. The purpose of this study was to investigate the molecular mechanism of hyperthyroidism-induced cardiomyopathy (HTC) and the effect of N-acetylcysteine (NAC), an ROS inhibitor, on the pathophysiology of HTC in vivo and in vitro. Compared with those in the control groups in vivo and in vitro, TT3 and TT4 were significantly increased, the structure of myocardial cells was enlarged and disordered, and interstitial fibrosis and the apoptosis of myocardial cells were markedly increased in the L-Thy group. The ROS and inflammatory response were increased in the hyperthyroidism group. In the NAC group, the contents of TT3 and TT4 were decreased, the myocardial cell structure was slightly disturbed, fibrosis and apoptosis were significantly reduced, and the ROS level and inflammatory response were significantly reduced. Interestingly, L-Thy decreased the viability of fibroblasts and H9c2 cells, suggesting that L-Thy-induced fibrosis was not caused by the proliferation of fibroblasts. The molecular mechanism of HTC could be explained by the fact that L-Thy could cause cardiac hypertrophy, inflammation, and fibrosis by regulating the Ca2+/calpain/Rcan1-dependent signalling pathway, the Ca2+/Rcan1/NF-κB/p65-dependent signalling pathway, and the Ca2+/ROS/Bcl-2/caspase-3-dependent signalling pathway. In conclusion, NAC can alleviate the pathophysiology of hyperthyroidism-induced cardiomyopathy, probably by regulating the ROS/Ca2+-dependent pathway.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are related neurodegenerative disorders displaying substantial overlay, although there are substantial differences at the molecular level. Currently, there is no effective treatment for these diseases. The transcription factor NRF2 has been postulated as a promising therapeutic target as it is capable of modulating key pathogenic events affecting cellular homeostasis. However, there is little experimental evidence on the status of this pathway in both ALS and FTD. Therefore, in this work, we wanted to carry out an exhaustive analysis of this signaling pathway in both transgenic mouse models (ALS and FTD) and human samples from patients with sporadic ALS (sALS) versus controls. In samples from patients with sALS and in the transgenic model with overexpression of TDP-43A315T, we observed a significant increase in the NRF2/ARE pathway in the motor cortex and the spinal cord, indicating that NRF2 antioxidant signaling was being induced, but it was not enough to reach cellular homeostasis. On the other hand, in the transgenic FTD model with overexpression of the TDP-43WT protein in forebrain neurons, a significantly decreased expression of NQO1 in the prefrontal cortex was seen, which cannot be attributed to alterations in the NRF2 pathway. Our results show that NRF2 signature is differently affected for ALS and FTD.

Recently, FGFR inhibitors have been highlighted as promising targeted drugs due to the high prevalence of FGFR1 amplification in cancer patients. Although various potential biomarkers for FGFR inhibitors have been suggested, their functional effects have been shown to be limited due to the complexity of the cancer signaling network and the heterogenous genomic conditions of patients. To overcome such limitations, we have reconstructed a lung cancer network model by integrating a cell line genomic database and analyzing the model in order to understand the underlying mechanism of heterogeneous drug responses. Here, we identify novel genomic context-specific candidates that can increase the efficacy of FGFR inhibitors. Furthermore, we suggest optimal targets that can induce more effective therapeutic responses than that of FGFR inhibitors in each of the FGFR-resistant lung cancer cells through computational simulations at a system level. Our findings provide new insights into the regulatory mechanism of differential responses to FGFR inhibitors for optimal therapeutic strategies in lung cancer.

Biosensor-integrated drug delivery systems are innovative devices in the health area, enabling continuous monitoring and drug administration. The use of smart polymer, bioMEMS, and electrochemical sensors have been extensively studied for these systems, especially for chronic diseases such as diabetes mellitus, cancer and cardiovascular diseases as well as advances in regenerative medicine. Basically, the technology involves sensors designed for the continuous analysis of biological molecules followed by drug release in response to specific signals. The advantages include high sensitivity and fast drug release. In this work, the main advances of biosensor-integrated drug delivery systems as new biomedical materials to improve the patients' quality of life with chronic diseases are discussed.

To examine whether the type of blood collection tubes affects the quantification of plasma biomarkers for Alzheimer's disease analyzed with a single-molecule array (Simoa), we recruited a healthy cohort (n = 34, 11 males, mean age = 28.7 ± 7.55) and collected plasma in the following tubes: dipotassium ethylenediaminetetraacetic acid (K2-EDTA), heparin lithium (Li-Hep), and heparin sodium (Na-Hep). Plasma tau, phosphorylated tau 181 (p-tau181), amyloid β (1-40) (Aβ40), and amyloid β (1-42) (Aβ42) were quantified using Simoa. We compared the value of plasma analytes, as well as the effects of sex on the measurements. We found that plasma collected in Li-Hep and Na-Hep tubes yielded significantly higher tau and p-tau181 levels compared to plasma collected in K2-EDTA tubes from the same person, but there was no difference in the measured values of the Aβ40, Aβ42, and Aβ42/40 ratio. Therefore, the type of blood collecting tubes should be considered when planning studies that measure plasma tau.

MicroRNAs (miRNAs) play an essential role in many biological processes. In this study, miRNAs in the skeletal muscle of normal and intrauterine growth retardation (IUGR) neonatal piglets were identified by sequencing, and canonical miRNAs were functionally validated in vitro. A total of 403 miRNAs were identified in neonatal piglet skeletal muscle, among them 30 and 46 miRNAs were upregulated and downregulated in IUGR pigs, respectively. Upregulated miRNAs were mainly enriched in propanoate metabolism, endocytosis, beta-Alanine metabolism, gap junction, and tumor necrosis factor signaling pathway. Down-regulated miRNAs were mainly enriched in chemical carcinogenesis-receptor activation, endocytosis, MAPK signaling pathway, insulin resistance, and EGFR tyrosine kinase inhibitor resistance. Co-expression network analysis of umbilical cord blood and skeletal muscle miRNAs showed that the miR-29 family is an essential regulator of IUGR pigs. The dual-luciferase reporter system showed that IGF1 and CCND1 were target genes of the miR-29 family. Transfection of IUGR pig umbilical cord blood exosomes and miR-29a mimic significantly inhibited cell proliferation and promoted the expression of cellular protein degradation marker genes Fbxo32 and Trim63. In summary, these results enrich the regulatory network of miRNAs involved in skeletal muscle development in IUGR animals.

Vulnerable plaques have been a hot topic in the field of stroke and carotid atherosclerosis. Currently, risk stratification and intervention of carotid plaques are guided by the degree of luminal stenosis. Recently, it has been recognized that the vulnerability of plaques may contribute to the risk of stroke. Some classical interventions, such as carotid endarterectomy, significantly reduce the risk of stroke in symptomatic patients with severe carotid stenosis, while for asymptomatic patients, clinically silent plaques with rupture tendency may expose them to the risk of cerebrovascular events. Early identification of vulnerable plaques contributes to lowering the risk of cerebrovascular events. Previously, the identification of vulnerable plaques was commonly based on imaging technologies at the macroscopic level. Recently, some microscopic molecules pertaining to vulnerable plaques have emerged, and could be potential biomarkers or therapeutic targets. This review aimed to update the previous summarization of vulnerable plaques and identify vulnerable plaques at the microscopic and macroscopic levels.

Palmitoylethanolamide (PEA), the naturally occurring amide of ethanolamine and palmitic acid, is an endogenous lipid compound endowed with a plethora of pharmacological functions, including analgesic, neuroprotective, immune-modulating, and anti-inflammatory effects. Although the properties of PEA were first characterized nearly 65 years ago, the identity of the receptor mediating these actions has long remained elusive, causing a period of research stasis. In the last two decades, a renewal of interest in PEA occurred, and a series of interesting studies have demonstrated the pharmacological properties of PEA and clarified its mechanisms of action. Recent findings showed the ability of formulations containing PEA in promoting oligodendrocyte differentiation, which represents the first step for the proper formation of myelin. This evidence opens new and promising research opportunities. White matter defects have been detected in a vast and heterogeneous group of diseases, including age-related neurodegenerative disorders. Here, we summarize the history and pharmacology of PEA and discuss its therapeutic potential in restoring white matter defects.

Matrix metalloproteinases (MMPs) are members of an enzyme family that are critical for maintaining tissue allostasis [...].

The cerebellum, or "little brain", is often overlooked in studies of brain metabolism in favour of the cortex. Despite this, anomalies in cerebellar amino acid homeostasis in a range of disorders have been reported. Amino acid homeostasis is central to metabolism, providing recycling of carbon backbones and ammonia between cell types. Here, we examined the role of cerebellar amino acid transporters in the cycling of glutamine and alanine in guinea pig cerebellar slices by inhibiting amino acid transporters and examining the resultant metabolism of [1-13C]d-glucose and [1,2-13C]acetate by NMR spectroscopy and LCMS. While the lack of specific inhibitors of each transporter makes interpretation difficult, by viewing results from experiments with multiple inhibitors we can draw inferences about the major cell types and transporters involved. In cerebellum, glutamine and alanine transfer is dominated by system A, blockade of which has maximum effect on metabolism, with contributions from System N. Inhibition of neural system A isoform SNAT1 by MeAIB resulted in greatly decreased metabolite pools and reduced net fluxes but showed little effect on fluxes from [1,2-13C]acetate unlike inhibition of SNAT3 and other glutamine transporters by histidine where net fluxes from [1,2-13C]acetate are reduced by ~50%. We interpret the data as further evidence of not one but several glutamate/glutamine exchange pools. The impact of amino acid transport inhibition demonstrates that the cerebellum has tightly coupled cells and that glutamate/glutamine, as well as alanine cycling, play a major role in that part of the brain.

There have been magnificent advancements in the understanding of molecular mechanisms of chronic diseases over the past several years, but these diseases continue to be a considerable cause of death worldwide. Most of the approved medications available for the prevention and treatment of these diseases target only a single gene/protein/pathway and are known to cause severe side effects and are less effective than they are anticipated. Consequently, the development of finer therapeutics that outshine the existing ones is far-reaching. Natural compounds have enormous applications in curbing several disastrous and fatal diseases. Oroxylin A (OA) is a flavonoid obtained from the plants Oroxylum indicum, Scutellaria baicalensis, and S. lateriflora, which have distinctive pharmacological properties. OA modulates the important signaling pathways, including NF-κB, MAPK, ERK1/2, Wnt/β-catenin, PTEN/PI3K/Akt, and signaling molecules, such as TNF-α, TGF-β, MMPs, VEGF, interleukins, Bcl-2, caspases, HIF-1α, EMT proteins, Nrf-2, etc., which play a pivotal role in the molecular mechanism of chronic diseases. Overwhelming pieces of evidence expound on the anti-inflammatory, anti-bacterial, anti-viral, and anti-cancer potentials of this flavonoid, which makes it an engrossing compound for research. Numerous preclinical and clinical studies also displayed the promising potential of OA against cancer, cardiovascular diseases, inflammation, neurological disorders, rheumatoid arthritis, osteoarthritis, etc. Therefore, the current review focuses on delineating the role of OA in combating different chronic diseases and highlighting the intrinsic molecular mechanisms of its action.

ALIAmides are a family of fatty acid amides whose name comes from their mechanism of action, i.e., the Autacoid Local Injury Antagonism (ALIA). Actually, the ALIAmide parent molecule, palmitoylethanolamide (PEA), is locally produced on demand from a cell membrane precursor in order to control immune-inflammatory cell responses, avert chronic non-resolving inflammation, and limit the resulting clinical signs. ALIAmide sister compounds, such as Adelmidrol and palmitoylglucosamine, share mechanisms of action with PEA and may also increase endogenous levels of PEA. Provided that their respective bioavailability is properly addressed (e.g., through decreasing the particle size through micronization), exogenously administered ALIAmides thus mimic or sustain the prohomeostatic functions of endogenous PEA. The aim of the present paper is to review the main findings on the use of ALIAmides in small animals as a tribute to the man of vision who first believed in this "according-to-nature" approach, namely Francesco della Valle. After briefly presenting some key issues on the molecular targets, metabolism, and pharmacokinetics of PEA and related ALIAmides, here we will focus on the preclinical and clinical studies performed in dogs and cats. Although more data are still needed, ALIAmides may represent a novel and promising approach to small animal health.

In this study, we examined the protective effects of curcumin against the AFB1-induced immune response of and pathological changes in broilers. Histopathology examinations showed that at day 28, AFB1 (5 mg/kg) exposure leads to severe histological changes in the spleen, thymus and bursa of Fabricius with a decrease in the number and karyoplasmic area ratio of plasma cells. Curcumin alleviated the AFB1-induced immune organs' damage as well as the changes in plasma cells in a dose-dependent manner. RT-PCR data showed that AFB1 significantly downregulated the IL-2 and IFN-γ mRNA expression levels in the thymus, spleen and bursa of Fabricius. However, curcumin supplementation improved the AFB1-induced immune organs' damage via upregulated cytokines' expression. Intriguingly, similar trends were noticed in abnormal morphological changes and the immune response at day 35 after the withdrawal of AFB1 and curcumin from the diet, suggesting the protective effects and immunomodulatory function against AFB1 in broilers. The current study provides a scientific experimental basis for the application of curcumin as a therapeutic drug or additive in animal husbandry productive practice.

The definition of the structural basis of the conformational preferences of the genetically encoded amino acid residues is an important yet unresolved issue of structural biology. In order to gain insights into this intricate topic, we here determined and compared the amino acid propensity scales for different (φ, ψ) regions of the Ramachandran plot and for different secondary structure elements. These propensities were calculated using the Chou-Fasman approach on a database of non-redundant protein chains retrieved from the Protein Data Bank. Similarities between propensity scales were evaluated by linear regression analyses. One of the most striking and unexpected findings is that distant regions of the Ramachandran plot may exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot may present anticorrelated propensities. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. The concept that similarities of the propensity scales are dictated by the similarity of the NCαC angle and not necessarily by the similarity of the (φ, ψ) conformation may have far-reaching implications in the field.

Single-stranded DNA (ssDNA) binding proteins (SSBs) are critical in maintaining genome stability by protecting the transient existence of ssDNA from damage during essential biological processes, such as DNA replication and gene transcription. The single-stranded region of telomeres also requires protection by ssDNA binding proteins from being attacked in case it is wrongly recognized as an anomaly. In addition to their critical roles in genome stability and integrity, it has been demonstrated that ssDNA and SSB-ssDNA interactions play critical roles in transcriptional regulation in all three domains of life and viruses. In this review, we present our current knowledge of the structure and function of SSBs and the structural features for SSB binding specificity. We then discuss the machine learning-based approaches that have been developed for the prediction of SSBs from double-stranded DNA (dsDNA) binding proteins (DSBs).

The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease.

Microglia, together with astrocytes and pericytes, cooperate to ensure blood-brain barrier (BBB) stability, modulating endothelial responses to inflammatory insults. Agonists of the sphingosine 1 phosphate (S1P) receptors, such as siponimod (BAF-312), are important pharmacological tools in multiple sclerosis and other inflammatory diseases. Modulation of S1P receptors may result in a reduced inflammatory response and increased BBB stability. An in vitro BBB model was reproduced using human-derived endothelial cells, astrocytes and microglia. Co-cultures were exposed to inflammatory cytokines (TNFα, 10 UI and IFNγ, 5 UI) in the presence of BAF-312 (100 nM), and the BBB properties and microglia role were evaluated. The drug facilitated microglial migration towards endothelial/astrocyte co-cultures, involving the activity of the metalloprotease 2 (MMP2). Microglia actively cooperated with astrocytes in the maintenance of endothelial barrier stability: in the triple co-culture, selective treatment of microglial cells with BAF-312 significantly prevented cytokines' effects on the endothelial barrier. In conclusion, BAF-312, modulating S1P receptors in microglia, may contribute to the reinforcement of the endothelial barrier at the BBB, suggesting an additional effect of the drug in the treatment of multiple sclerosis.

Olive oil is considered to be a food of utmost importance, especially in the Mediterranean countries. The quality of olive oil must remain stable regarding authenticity and storage. This review paper emphasizes the detection of olive oil oxidation status or rancidity, the analytical techniques that are usually used, as well as the application and significance of chemometrics in the research of olive oil. The first part presents the effect of the oxidation of olive oil during storage. Then, lipid stability measurements are described in parallel with instrumentation and different analytical techniques that are used for this particular purpose. The next part presents some research publications that combine chemometrics and the study of lipid changes due to storage published in 2005-2021. Parameters such as exposure to light, air and various temperatures as well as different packaging materials were investigated to test olive oil stability during storage. The benefits of each chemometric method are provided as well as the overall significance of combining analytical techniques and chemometrics. Furthermore, the last part reflects on fraud in olive oil, and the most popular analytical techniques in the authenticity field are stated to highlight the importance of the authenticity of olive oil.

Complications to preterm birth are numerous, including the presence of a patent ductus arteriosus (PDA). The biological understanding of the PDA is sparse and treatment remains controversial. Herein, we speculate whether the PDA is more than a cardiovascular imbalance, and may be a marker in response to immature core molecular and physiological processes driven by biological systems, such as inflammation. To achieve a new biological understanding of the PDA, we performed echocardiography and collected plasma samples on day 3 of life in 53 consecutively born neonates with a gestational age at birth below 28 completed weeks. The proteome of these samples was analyzed by mass spectrometry (nanoLC-MS/MS) and immunoassay of 17 cytokines and chemokines. We found differences in 21 proteins and 8 cytokines between neonates with a large PDA (>1.5 mm) compared to neonates without a PDA. Amongst others, we found increased levels of angiotensinogen, periostin, pro-inflammatory associations, including interleukin (IL)-1β and IL-8, and anti-inflammatory associations, including IL-1RA and IL-10. Levels of complement factors C8 and carboxypeptidases were decreased. Our findings associate the PDA with the renin-angiotensin-aldosterone system and immune- and complement systems, indicating that PDA goes beyond the persistence of a fetal circulatory connection of the great vessels.

Butyrate is produced in the rumen from microbial fermentation and is related to several functions, including cell differentiation and proliferation. Butyrate supplementation in calves can accelerate rumen development. DNA-protein interactions, such as the CCCTC-binding factor (CTCF), play essential roles in chromatin organization and gene expression regulation. Although CTCF-binding sites have been identified recently in cattle, a deeper characterization, including differentially CTCF-binding sites (DCBS), is vital for a better understanding of butyrate's role in the chromatin landscape. This study aimed to identify CTCF-binding regions and DCBS under a butyrate-induced condition using ChIP-seq in bovine cells; 61,915 CTCF peaks were identified in the butyrate and 51,347 in the control. From these regions, 2265 DCBS were obtained for the butyrate vs. control comparison, comprising ~90% of induced sites. Most of the butyrate DCBS were in distal intergenic regions, showing a potential role as insulators. Gene ontology enrichment showed crucial terms for the induced DCBS, mainly related to cellular proliferation, cell adhesion, and growth regulation. Interestingly, the ECM-receptor interaction pathway was observed for the induced DCBS. Motif enrichment analysis further identified transcription factors, including CTCF, BORIS, TGIF2, and ZIC3. When DCBS was integrated with RNA-seq data, putative genes were identified for the repressed DCBS, including GATA4. Our study revealed promising candidate genes in bovine cells by a butyrate-induced condition that might be related to the regulation of rumen development, such as integrins, keratins, and collagens. These results provide a better understanding of the function of butyrate in cattle rumen development and chromatin landscape regulation.

Sodium glucose cotransporter, type 2 inhibitors, such as Empagliflozin, are protective of the kidneys by unclear mechanisms. Our aim was to determine how Empagliflozin affected kidney cortical metabolome and lipidome in mice. Adult male TALLYHO mice (prone to obesity) were treated with a high-milk-fat diet, or this diet containing Empagliflozin (0.01%), for 8 weeks. Targeted and untargeted metabolomics and lipidomics were conducted on kidney cortex by liquid chromatography followed by tandem mass-spectroscopy. Metabolites were statistically analyzed by MetaboAnalyst 5.0, LipidSig (lipid species only) and/or CEU Mass Mediator (untargeted annotation). In general, volcano plotting revealed oppositely skewed patterns for targeted metabolites (primarily hydrophilic) and lipids (hydrophobic) in that polar metabolites showed a larger number of decreased species, while non-polar (lipids) had a greater number of increased species (>20% changed and/or raw p-value < 0.05). The top three pathways regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism, with an amino acid network being highly affected, with 14 of 20 classic amino acids down-regulated. Out of 75 changed polar metabolites, only three were up-regulated, i.e., flavin mononucleotide (FMN), uridine, and ureidosuccinic acid. Both FMN and uridine have been shown to be protective of the kidney. Scrutiny of metabolites of glycolysis/gluconeogenesis/Krebs cycle revealed a 20-45% reduction in several species, including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, although overall lipid quantity was not higher, several lipid species were increased by EMPA, including those of the classes, phosphatidic acids, phosphatidylcholines, and carnitines. Overall, these analyses suggest a protection from extensive metabolic load and the corresponding oxidative stress with EMPA in kidney. This may be in response to reduced energy demands of the proximal tubule as a result of inhibition of transport and/or differences in metabolic pools available for metabolism.

Malate dehydrogenase (MDH), which catalyzes a reversible conversion of L-malate to oxaloacetate, plays essential roles in common metabolic processes, such as the tricarboxylic acid cycle, the oxaloacetate-malate shuttle, and the glyoxylate cycle. MDH2 has lately been recognized as a promising anticancer target; however, the structural information for the human homologue with natural ligands is very limited. In this study, various complex structures of hMDH2, with its substrates and/or cofactors, were solved by X-ray crystallography, which could offer knowledge about the molecular and enzymatic mechanism of this enzyme and be utilized to design novel inhibitors. The structural comparison suggests that phosphate binds to the substrate binding site and brings the conformational change of the active loop to a closed state, which can secure the substate and cofactor to facilitate enzymatic activity.

Breast cancer (BC) is one of the most common types of malignancies in women and greatly threatens female health. KRT17 is a member of the keratin (KRT) protein family that is abundant in the outer layer of the skin, where it protects epithelial cells from damage. Although KRT17 has been studied in many types of cancer, the expression of KRT17 in specific subtypes of BC remains to be determined. In our study, we explored the expression and prognostic implications of KRT17 in BC patients using mRNA transcriptome data and clinical BC data from The Cancer Genome Atlas (TCGA). Receiver operating characteristic (ROC) curves and the chi-square test were used to assess the diagnostic value of KRT17 expression. Quantitative real-time PCR (qRT-PCR) analysis of BC cells and tissues and immunohistochemistry (IHC) analysis of clinical tissues were used for external validation. Furthermore, the relationship between KRT17 and immune function was studied by using the CIBERSORT algorithm to predict the proportions of tumor-infiltrating immune cells (TIICs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the potential mechanisms by which KRT17 expression influences patient survival. We found that KRT17 expression was significantly lower in BC tissues than in normal tissues, especially in the luminal-A, luminal-B and human epidermal growth factor receptor-2 (HER2)+ subtypes of BC. ROC analysis revealed that KRT17 expression had moderate diagnostic value. Interestingly, decreased expression of KRT17 was significantly correlated with poor prognosis in BC patients, especially in HER2high and ERhigh patients. This trend was also verified by tissue microarray (TMA) analysis. KRT17 was found to be involved in some antitumor immune pathways, especially the IL-17 signaling pathway, and associated with multiple immune cells, such as natural killer (NK) and CD4+ T cells. In conclusion, high expression of KRT17 predicted favorable prognosis in BC patients with higher HER2 expression. This result may indicate that KRT17 plays a different role depending on the level of HER2 expression and could serve as a promising and sensitive biomarker for the diagnosis and prognostication of HER2high BC.

Nowadays, T-cell-based approaches play an increasing role in cancer treatment. In particular, the use of (genetically engineered) T-cells has heralded a novel era for various diseases with previously poor outcomes. Concurrently, the relationship between the functional behavior of immune cells and their metabolic state, known as immunometabolism, has been found to be an important determinant for the success of immunotherapy. In this context, immune cell metabolism is not only controlled by the expression of transcription factors, enzymes and transport proteins but also by nutrient availability and the presence of intermediate metabolites. The lack of as well as an oversupply of nutrients can be detrimental and lead to cellular dysfunction and damage, potentially resulting in reduced metabolic fitness and/or cell death. This review focusses on the detrimental effects of excessive exposure of T cells to fatty acids, known as lipotoxicity, in the context of an altered lipid tumor microenvironment. Furthermore, implications of T cell-related lipotoxicity for immunotherapy will be discussed, as well as potential therapeutic approaches.

Inonotus obliquus grows in the Northern Hemisphere on some living broadleaved tree species as a pathogen, causing stem rot. In Estonia, the fungus is well known in the Betula species but can also be found on Alnus. Sterile conks of I. obliquus contain different bioactive compounds, but the quantitative and comparative research of these compounds in conks on different host species is limited. In the current work, I. obliquus was isolated and, evidently, determined from Alnus incana (L.) Moench., Alnus glutinosa (L.) Gaertn., and Betula pendula Roth, and the content of bioactive compounds in conks on these hosts were analysed. All the analysed conks sampled from A. incana and B. pendula contained betulin that varied from 111 to 159 µg/g. A significantly (p < 0.05) higher betulinic acid content was found in conks sampled from A. incana when compared with B. pendula: 474-635 and 20-132 µg/g, respectively. However, the conks from Betula were richer in total polyphenols, flavonols, and glucans. The content of inotodiol was quite similar in the conks from A. incana (7455-8961 µg/g) and B. pendula (7881-9057 µg/g). Also, no significant differences in the lanosterol content were found between the samples from these two tree species. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on Alnus. The results demonstrate that the bioactive compounds are promising in conks of I. obliquus growing not only on Betula but also on the Alnus species. It supports the opportunity to cultivate I. obliquus, also on the Alnus species, thus increasing the economic value of growing this tree species in forestry.

Heme proteins serve diverse and pivotal biological functions. Therefore, clarifying the mechanisms of these diverse functions of heme is a crucial scientific topic. Distortion of heme porphyrin is one of the key factors regulating the chemical properties of heme. Here, we constructed convolutional neural network models for predicting heme distortion from the tertiary structure of the heme-binding pocket to examine their correlation. For saddling, ruffling, doming, and waving distortions, the experimental structure and predicted values were closely correlated. Furthermore, we assessed the correlation between the cavity shape and molecular structure of heme and demonstrated that hemes in protein pockets with similar structures exhibit near-identical structures, indicating the regulation of heme distortion through the protein environment. These findings indicate that the tertiary structure of the heme-binding pocket is one of the factors regulating the distortion of heme porphyrin, thereby controlling the chemical properties of heme relevant to the protein function; this implies a structure-function correlation in heme proteins.

Gastrointestinal (GI) neoplasms include esophageal, gastric, colorectal, hepatic, and pancreatic cancers. They are characterized by asymptomatic behavior, being responsible for diagnostic delay. Substantial refractoriness to chemo- and radiotherapy, exhibited by late-stage tumors, contribute to determine poor patient outcome. Therefore, it is of outmost importance to identify new molecular targets for the development of effective therapeutic strategies. In this study, we focused on the enzyme nicotinamide N-methyltransferase (NNMT), which catalyzes the N-methylation reaction of nicotinamide and whose overexpression has been reported in numerous neoplasms, including GI cancers. The aim of this review was to report data illustrating NNMT involvement in these tumors, highlighting its contribution to tumor cell phenotype. Cited works clearly demonstrate the interesting potential use of enzyme level determination for both diagnostic and prognostic purposes. NNMT was also found to positively affect cell viability, proliferation, migration, and invasiveness, contributing to sustain in vitro and in vivo tumor growth and metastatic spread. Moreover, enzyme upregulation featuring tumor cells was significantly associated with enhancement of resistance to treatment with chemotherapeutic drugs. Taken together, these results strongly suggest the possibility to target NNMT for setup of molecular-based strategies to effectively treat GI cancers.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

Extracellular vesicles (EVs), which are nanosized membranous particles secreted from both prokaryotic and eukaryotic cells, can deliver various biological molecules, such as nucleic acids, proteins, and lipids, into recipient cells. However, contrary to what is known about eukaryotic EVs, whether bacterial EVs (bEVs) can be used as transporters for bioactive molecules is becoming a hot area of research. In this study, we electroporated enhanced green fluorescent protein (EGFP) genes and precursor microRNA of Cel-miR-39 (pre-Cel-miR-39) from isolated bEVs of Escherichia coli and Lactobacillus reuteri. The EGFP plasmid, synthetic EGFP RNA, and pre-Cel-miR-39 were successfully delivered into the murine microglial BV2 cells via bEVs. PCR and confocal microscopy analysis confirmed the transfer of the EGFP plasmid and RNA. The bEV-delivered exogenous pre-Cel-miR-39 was further processed into the mature form of Cel-miR-39; its incorporation into Ago2-a major component of the RNA-induced silencing complex-was assessed using RNA-immunoprecipitation-PCR. Taken together, bEVs can be used as vehicles to deliver genetic materials and for novel biotechnological applications, such as gene transfer and mRNA vaccines.

The brain renin-angiotensin system plays important roles in blood pressure and cardiovascular regulation. There are two isoforms of prorenin in the brain: the classic secreted form (prorenin/sREN) encoded by renin-a, and an intracellular form (icREN) encoded by renin-b. Emerging evidence indicates the importance of renin-b in cardiovascular and metabolic regulation. However, the role of endogenous brain prorenin in the development of salt-sensitive hypertension remains undefined. In this study, we test the hypothesis that renin-a produced locally in the brain contributes to the pathogenesis of hypertension. Using RNAscope, we report for the first time that renin mRNA is expressed in several regions of the brain, including the subfornical organ (SFO), the paraventricular nucleus of the hypothalamus (PVN), and the brainstem, where it is found in glutamatergic, GABAergic, cholinergic, and tyrosine hydroxylase-positive neurons. Notably, we found that renin mRNA was significantly elevated in the SFO and PVN in a mouse model of DOCA-salt-induced hypertension. To examine the functional importance of renin-a in the SFO, we selectively ablated renin-a in the SFO in renin-a-floxed mice using a Cre-lox strategy. Importantly, renin-a ablation in the SFO attenuated the maintenance of DOCA-salt-induced hypertension and improved autonomic function without affecting fluid or sodium intake. Molecularly, ablation of renin-a prevented the DOCA-salt-induced elevation in NADPH oxidase 2 (NOX2) in the SFO without affecting NOX4 or angiotensin II type 1 and 2 receptors. Collectively, our findings demonstrate that endogenous renin-a within the SFO is important for the pathogenesis of salt-sensitive hypertension.

Microcrystalline cellulose (MCC) is an emerging material with outstanding properties in many scientific and industrial fields, in particular as an additive in composite materials. Its surface modification allows for the fine-tuning of its properties and the exploitation of these materials in a plethora of applications. In this paper, we present the covalent linkage of a luminescent Ir-complex onto the surface of MCC, representing the first incorporation of an organometallic luminescent probe in this biomaterial. This goal has been achieved with an easy and sustainable procedure, which employs a Bronsted-acid ionic liquid as a catalyst for the esterification reaction of -OH cellulose surface groups. The obtained luminescent cellulose microcrystals display high and stable emissions with the incorporation of only a small amount of iridium (III). Incorporation of MCC-Ir in dry and wet matrices, such as films and gels, has been also demonstrated, showing the maintenance of the luminescent properties even in possible final manufacturers.

Tau pathology extends throughout the brain in a prion-like fashion through connected brain regions. However, the details of the underlying mechanisms are incompletely understood. The present study aims to examine the spreading of P301S aggregated tau, a mutation that is implicated in tauopathies, using organotypic slice cultures. Coronal hippocampal organotypic brain slices (170 µm) were prepared from postnatal (day 8-10) C57BL6 wild-type mice. Collagen hydrogels loaded with P301S aggregated tau were applied to slices and the spread of tau was assessed by immunohistochemistry after 8 weeks in culture. Collagen hydrogels prove to be an effective protein delivery system subject to natural degradation in 14 days and they release tau proteins up to 8 weeks. Slices with un- and hyperphosphorylated P301S aggregated tau demonstrate significant spreading to the ventral parts of the hippocampal slices compared to empty collagen hydrogels after 8 weeks. Moreover, the spread of P301S aggregated tau occurs in a time-dependent manner, which was interrupted when the neuroanatomical pathways are lesioned. We illustrate that the spreading of tau can be investigated in organotypic slice cultures using collagen hydrogels to achieve a localized application and slow release of tau proteins. P301S aggregated tau significantly spreads to the ventral areas of the slices, suggesting that the disease-relevant aggregated tau form possesses spreading potential. Thus, the results offer a novel experimental approach to investigate tau pathology.

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14+CD16- monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes.

Radiotherapy remains an effective conventional method of treatment for patients with cancer. However, the clinical efficacy of radiotherapy is compromised by the development of radioresistance of the tumor cells during the treatment. Consequently, there is need for a comprehensive understanding of the regulatory mechanisms of tumor cells in response to radiation to improve radiotherapy efficacy. The current study aims to highlight new developments that illustrate various forms of cancer cell death after exposure to radiation. A summary of the cellular pathways and important target proteins that are responsible for tumor radioresistance and metastasis is also provided. Further, the study outlines several mechanistic descriptions of the interaction between ionizing radiation and the host immune system. Therefore, the current review provides a reference for future research studies on the biological effects of new radiotherapy technologies, such as ultra-high-dose-rate (FLASH) radiotherapy, proton therapy, and heavy-ion therapy.

The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β. These two isoforms are 85% identical and encoded by two different genes. Hsp90β is constitutively expressed and essential for early mouse development, while Hsp90α is stress-inducible and not necessary for survivability. These two isoforms are known to have largely overlapping functions and to interact with a large fraction of the proteome. To what extent there are isoform-specific functions at the protein level has only relatively recently begun to emerge. There are studies indicating that one isoform is more involved in the functionality of a specific tissue or cell type. Moreover, in many diseases, functionally altered cells appear to be more dependent on one particular isoform. This leaves space for designing therapeutic strategies in an isoform-specific way, which may overcome the unfavorable outcome of pan-Hsp90 inhibition encountered in previous clinical trials. For this to succeed, isoform-specific functions must be understood in more detail. In this review, we summarize the available information on isoform-specific functions of mammalian Hsp90 and connect it to possible clinical applications.

Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy.

Increasing evidence strongly supports the key role of neuroinflammation in the pathophysiology of neurodegenerative diseases, such as Alzheimer's disease, frontotemporal dementia, and amyotrophic lateral sclerosis. Neuroinflammation may alter synaptic transmission contributing to the progression of neurodegeneration, as largely documented in animal models and in patients' studies. In the last few years, palmitoylethanolamide (PEA), an endogenous lipid mediator, and its new composite, which is a formulation constituted of PEA and the well-recognized antioxidant flavonoid luteolin (Lut) subjected to an ultra-micronization process (co-ultraPEALut), has been identified as a potential therapeutic agent in different disorders by exerting potential beneficial effects on neurodegeneration and neuroinflammation by modulating synaptic transmission. In this review, we will show the potential therapeutic effects of PEA in animal models and in patients affected by neurodegenerative disorders.

Normal function of placental extravillous trophoblasts (EVTs), which are responsible for uteroplacental vascular remodeling, is critical for adequate delivery of oxygen and nutrients to the developing fetus and normal fetal programming. Proliferation and invasion of spiral arteries by EVTs depends upon adequate levels of folate. Multidrug resistance-associated protein 1 (MRP1), which is an efflux transporter, is known to remove folate from these cells. We hypothesized that palmitic acid increases MRP1-mediated folate removal from EVTs, thereby interfering with EVTs' role in early placental vascular remodeling. HTR-8/SVneo and Swan-71 cells, first trimester human EVTs, were grown in the absence or presence of 0.5 mM and 0.7 mM palmitic acid, respectively, for 72 h. Palmitic acid increased ABCC1 gene expression and MRP1 protein expression in both cell lines. The rate of folate efflux from the cells into the media increased with a decrease in migration and invasion functions in the cultured cells. Treatment with N-acetylcysteine (NAC) prevented the palmitic acid-mediated upregulation of MRP1 and restored invasion and migration in the EVTs. Finally, in an ABCC1 knockout subline of Swan-71 cells, there was a significant increase in invasion and migration functions. The novel finding in this study that palmitic acid increases MRP1-mediated folate efflux provides a missing link that helps to explain how maternal consumption of saturated fatty acids compromises the in utero environment.

The aim of this study was to investigate the effects of the androgenic hormone testosterone enanthate (TE) on human MG-63 cells. MG-63 were cultured for 24 h in the presence of TE at increasing concentrations to assess its lethal dose. Therefore, the suitable concentration for a prolonged use of TE in vitro was assessed by viability assay over 9 days. Finally, MG-63 were exposed to TE for 14 days and assayed for differentiation by qPCR and Alizarin Red S staining. TE in the amount of 100 µM resulted as the maximum dose tolerated by MG-63 cells after 24 h. However, a prolonged exposure in culture TE in the amount of 100 µM showed a cytostatic effect on cell proliferation. On the contrary, TE 10 µM was tolerated by the cells and did not boost cell proliferation, but did enhance new bone formation, as revealed by COL1A1, ALPL, BGLAP, and IBSP gene expression after 3, 7, and 14 days, and calcium deposition by Alizarin Red S staining after 14 days. Based on the current study, 10 µM is the critical dose of TE that should be used in vitro to support bone differentiation of MG-63 cells.

Medial vascular calcification is common in chronic kidney disease (CKD) and is closely linked to hyperphosphatemia. Vascular smooth muscle cells (VSMCs) can take up pro-calcific properties and actively augment vascular calcification. Various pro-inflammatory mediators are able to promote VSMC calcification. In this study, we investigated the effects and mechanisms of periostin, a matricellular signaling protein, in calcifying human VSMCs and human serum samples. As a result, periostin induced the mRNA expression of pro-calcific markers in VSMCs. Furthermore, periostin augmented the effects of β-glycerophosphate on the expression of pro-calcific markers and aggravated the calcification of VSMCs. A periostin treatment was associated with an increased β-catenin abundance as well as the expression of target genes. The pro-calcific effects of periostin were ameliorated by WNT/β-catenin pathway inhibitors. Moreover, a co-treatment with an integrin αvβ3-blocking antibody blunted the pro-calcific effects of periostin. The silencing of periostin reduced the effects of β-glycerophosphate on the expression of pro-calcific markers and the calcification of VSMCs. Elevated serum periostin levels were observed in hemodialysis patients compared with healthy controls. These observations identified periostin as an augmentative factor in VSMC calcification. The pro-calcific effects of periostin involve integrin αvβ3 and the activation of the WNT/β-catenin pathway. Thus, the inhibition of periostin may be beneficial to reduce the burden of vascular calcification in CKD patients.

The outbreak of COVID-19 caused millions of deaths worldwide, and the number of total infections is still rising. It is necessary to identify some potentially effective drugs that can be used to prevent the development of severe symptoms, or even death for those infected. Fortunately, many efforts have been made and several effective drugs have been identified. The rapidly increasing amount of data is of great help for training an effective and specific deep learning model. In this study, we propose a multi-task deep learning model for the purpose of screening commercially available and effective inhibitors against SARS-CoV-2. First, we pretrained a model on several heterogenous protein-ligand interaction datasets. The model achieved competitive results on some benchmark datasets. Next, a coronavirus-specific dataset was collected and used to fine-tune the model. Then, the fine-tuned model was used to select commercially available drugs against SARS-CoV-2 protein targets. Overall, twenty compounds were listed as potential inhibitors. We further explored the model interpretability and exhibited the predicted important binding sites. Based on this prediction, molecular docking was also performed to visualize the binding modes of the selected inhibitors.

The aim of this study was to quantify ESKAPEE bacteria, genes encoding resistance to antibiotics targeting this group of pathogens, as well as integrase genes in municipal wastewater and river water. Environmental DNA was extracted from the collected samples and used in deep sequencing with the Illumina TruSeq kit. The abundance of bacterial genera and species belonging to the ESKAPEE group, 400 ARGs associated with this microbial group, and three classes of integrase genes were determined. A taxonomic analysis revealed that Acinetobacter was the dominant bacterial genus, whereas Acinetobacter baumannii and Escherichia coli were the dominant bacterial species. The analyzed samples were characterized by the highest concentrations of the following ARGs: blaGES, blaOXA-58, blaTEM, qnrB, and qnrS. Acinetobacter baumannii, E. coli, and genes encoding resistance to β-lactams (blaVEB-1, blaIMP-1, blaGES, blaOXA-58, blaCTX-M, and blaTEM) and fluoroquinolones (qnrS) were detected in samples of river water collected downstream from the wastewater discharge point. The correlation analysis revealed a strong relationship between A. baumannii (bacterial species regarded as an emerging human pathogen) and genes encoding resistance to all tested groups of antimicrobials. The transmission of the studied bacteria (in particular A. baumannii) and ARGs to the aquatic environment poses a public health risk.

Glial cells are the most abundant and widely distributed cells that maintain cerebral homeostasis in the central nervous system. They mainly include microglia, astrocytes, and the oligodendrocyte lineage cells. Moreover, glial cells may induce pathological changes, such as inflammatory responses, demyelination, and disruption of the blood-brain barrier, to regulate the occurrence and development of neurological diseases through various molecular mechanisms. Furthermore, RNA m6A modifications are involved in various pathological processes associated with glial cells. In this review, the roles of glial cells in physiological and pathological states, as well as advances in understanding the mechanisms by which glial cells regulate neurological diseases under RNA m6A modification, are summarized, hoping to provide new perspectives on the deeper mechanisms and potential therapeutic targets for neurological diseases.

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages' tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

Anther dehiscence is an important process to release pollen and then is a critical event in pollination. In the wheat photo-thermo-sensitive genic male sterility (PTGMS) line, pollen cannot release from anther since the anther cannot dehisce during anther dehiscence stage in a sterile condition. In this study, we carried out RNA-sequencing to analyze the transcriptome of one wheat PTGMS line BS366 during anther dehiscence under fertile and sterile conditions to explore the mechanism. We identified 6306 differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) and KEGG analysis showed that DEGs were mainly related to "hormone signal transduction pathway" and "starch and sucrose metabolism". We identified 35 and 23 DEGs related hormone signal transduction and sucrose metabolism, respectively. Compared with conventional wheat Jing411, there were some changes in the contents of hormones, including JA, IAA, BR, ABA and GA3, and sucrose, during three anther dehiscence stages in the sterile condition in BS366. We performed qRT-PCR to verify the expression levels of some critical DEGs of the hormone signaling pathway and the starch and sucrose metabolism pathway. The results showed disparate expression patterns of the critical DEGs of the hormone signaling pathway and the starch and sucrose metabolism pathway in different conditions, suggesting these genes may be involved in the regulation of the anther dehiscence in BS366. Finally, we conducted a hypothesis model to reveal the regulation pathway of hormones and sucrose on anther dehiscence. The information provided new clues to the molecular mechanisms of anther dehiscence in wheat and improved wheat hybrid breeding.

Mycobacterium tuberculosis, the causal agent of one of the most devastating infectious diseases worldwide, can evade or modulate the host immune response and remain dormant for many years. In this review, we focus on identifying the local immune response induced in vivo by M. tuberculosis in the lungs of patients with active tuberculosis by analyzing data from untouched cells from bronchoalveolar lavage fluid (BALF) or exhaled breath condensate (EBC) samples. The most abundant resident cells in patients with active tuberculosis are macrophages and lymphocytes, which facilitate the recruitment of neutrophils. The cellular response is characterized by an inflammatory state and oxidative stress produced mainly by macrophages and T lymphocytes. In the alveolar microenvironment, the levels of cytokines such as interleukins (IL), chemokines, and matrix metalloproteinases (MMP) are increased compared with healthy patients. The production of cytokines such as interferon (IFN)-γ and IL-17 and specific immunoglobulin (Ig) A and G against M. tuberculosis indicate that the adaptive immune response is induced despite the presence of a chronic infection. The role of epithelial cells, the processing and presentation of antigens by macrophages and dendritic cells, as well as the role of tissue-resident memory T cells (Trm) for in situ vaccination remains to be understood.

Over a hundred different autoimmune diseases have been described to date, which can affect every organ in the body, including the largest one, the skin. In fact, up to one-fifth of the world's population suffers from chronic, noninfectious inflammatory skin diseases, the development of which is significantly influenced by an autoimmune response. One of the hallmarks of autoimmune diseases is the loss of immune tolerance, which leads to the formation of autoreactive lymphocytes or autoantibodies and, consequently, to chronic inflammation and tissue damage. The treatment of autoimmune skin diseases mainly focuses on immunosuppression (using, e.g., corticosteroids) but almost never leads to the development of permanent mechanisms of immune tolerance. In addition, current therapies and their long-term administration may cause serious adverse effects. Hence, safer and more effective therapies that bring sustained balance between pro- and anti-inflammatory responses are still desired. Both intra- and extracellular heat shock proteins (Hsps), specifically well-characterized inducible Hsp90 and Hsp70 chaperones, have been highlighted as therapeutic targets for autoimmune diseases. This review presents preclinical data on the involvement of Hsp90 and Hsp70 in modulating the immune response, specifically in the context of the treatment of selected autoimmune skin diseases with emphasis on autoimmune bullous skin diseases and psoriasis.

Proteasomes comprise a family of proteasomal complexes essential for maintaining protein homeostasis. Accordingly, proteasomes represent promising therapeutic targets in multiple human diseases. Several proteasome inhibitors are approved for treating hematological cancers. However, their side effects impede their efficacy and broader therapeutic applications. Therefore, understanding the biology of the different proteasome complexes present in the cell is crucial for developing tailor-made inhibitors against specific proteasome complexes. Here, we will discuss the structure, biology, and function of the alternative Proteasome Activator 200 (PA200), also known as PSME4, and summarize the current evidence for its dysregulation in different human diseases. We hereby aim to stimulate research on this enigmatic proteasome regulator that has the potential to serve as a therapeutic target in cancer.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common complication of antineoplastic drugs, particularly paclitaxel (PTX). It can affect the quality of patients' lives and increase the risk of developing mood disorders. Although several drugs are recommended, they yielded inconclusive results in clinical trials. The aim of the present work is to investigate whether the palmitoylethanolamide (PEA) would reduce PTX-induced CIPN and associated mood disorders. Moreover, the role PPAR-α and the endocannabinoid system will also be investigated. CIPN was induced by intraperitoneally injection of PTX (8 mg/kg) every other day for a week. PEA, 30 mg/kg, was orally administrated in a bioavailable form (i.e., ultramicronized PEA, um-PEA) one hour after the last PTX injection, for 7 days. In the antagonism experiments, AM281 (1 mg/kg) and GW6471 (2 mg/kg) were administrated 30 min before um-PEA. Our results demonstrated that um-PEA reduced the development of hypersensitivity with the effect being associated with the reduction in spinal and hippocampal pro-inflammatory cytokines, as well as antidepressive and anxiolytic effects. Moreover, the PPAR-α and CB1 receptor antagonists blocked the behavioral and antinociceptive effects of um-PEA. Our findings suggest that um-PEA is a promising adjunct in CIPN and associated mood disorders through the activation of PPAR-α, which influences the endocannabinoid system.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.

α-Klotho (Klotho) is an antiaging hormone with anti-inflammatory and antioxidative properties. Some studies suggest that Klotho increases in response to enhanced oxidative damage and inflammation. Alcoholism is a proinflammatory condition. The aim of this study was to analyze the relationship between Klotho and the serum levels of the inflammatory markers in alcoholic liver disease and to assess its prognostic value. We included 184 alcoholics and 35 age- and sex-matched controls. We determined the serum levels of Klotho, the tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and malondialdehyde (MDA), and routine laboratory variables. Patients were followed-up with during 16 ± 18 months; 67 patients died. Klotho levels were higher among cirrhotics (with KW = 37.00 and p < 0.001) and were related to the Child-Pugh score (with KW = 15.96 and p < 0.001) and to the TNF-α (ρ = 0.28; p < 0.001) and MDA (ρ = 0.21; p = 0.006). The child's groups were associated with mortality, both in the univariate (with the log-rank = 13.56, p = 0.001, Breslow = 12.33, and p = 0.002) and multivariate (with β = 0.43, p = 0.02, and OR = 1.53 (1.07-2.15)) analyses, also introducing Klotho and the TNF-α as dichotomic variables. However, the independent prognostic value of the Child's groups was displaced by Klotho when only cirrhotics were considered; Klotho, over the median (574.4 pg/mL), was associated with higher mortality (with p = 0.04 and OR = 2.68 (1.06-6.84)). We conclude that Klotho is increased in liver cirrhosis. It is directly related to TNF-α, MDA, and to mortality in cirrhotics.

The abnormal regulation and expression of microRNA (miRNA) are closely related to the aging process and the occurrence and development of aging-related diseases. Lethal-7 (let-7) was discovered in Caenorhabditis elegans (C. elegans) and plays an important role in development by regulating cell fate regulators. Accumulating evidence has shown that let-7 is elevated in aging tissues and participates in multiple pathways that regulate the aging process, including affecting tissue stem cell function, body metabolism, and various aging-related diseases (ARDs). Moreover, recent studies have found that let-7 plays an important role in the senescence of B cells, suggesting that let-7 may also participate in the aging process by regulating immune function. Therefore, these studies show the diversity and complexity of let-7 expression and regulatory functions during aging. In this review, we provide a detailed overview of let-7 expression regulation as well as its role in different tissue aging and aging-related diseases, which may provide new ideas for enriching the complex expression regulation mechanism and pathobiological function of let-7 in aging and related diseases and ultimately provide help for the development of new therapeutic strategies.

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -β and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-β and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-β reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses.

The COVID-19 pandemic has been a major public health event since 2020. Multiple variant strains of SARS-CoV-2, the causative agent of COVID-19, were detected based on the mutation sites in their sequences. These sequence mutations may lead to changes in the protein structures and affect the binding states of SARS-CoV-2 and human proteins. Experimental research on SARS-CoV-2 has accumulated a large amount of structural data and protein-protein interactions (PPIs), but the studies on the SARS-CoV-2-human PPI networks lack integration of physical associations with possible protein docking information. In addition, the docking structures of variant viral proteins with human receptor proteins are still insufficient. This study constructed SARS-CoV-2-human protein-protein interaction network with data integration methods. Crystal structures were collected to map the interaction pairs. The pairs of direct interactions and physical associations were selected and analyzed for variant docking calculations. The study examined the structures of spike (S) glycoprotein of variants Delta B.1.617.2, Omicron BA.1, and Omicron BA.2. The calculated docking structures of S proteins and potential human receptors were obtained. The study integrated binary protein interactions with 3D docking structures to fulfill an extended view of SARS-CoV-2 proteins from a macro- to micro-scale.

Diabetic nephropathy is currently the leading cause of end-stage renal disease (ESRD) in type 2 diabetes. Studies have suggested that supplementation with some fatty acids might reduce the risk and delay the progression to ESRD in patient with chronic kidney disease. Crocodile oil (CO) contains a variety of fatty acids, especially omega-3, -6 and -9, that have been reported to be beneficial to human health. This study examined the impact of long-term CO supplementation on the development of diabetic nephropathy in spontaneously diabetic Torii (SDT) rats. After diabetic verification, SDT rats were assigned to receive vehicle or CO at 500 and 1000 mg/kg BW, respectively, by oral gavage. Age-matched nondiabetic Sprague-Dawley rats were given vehicle or high-dose CO. After 28 weeks of intervention, CO failed to improve hyperglycemia and pancreatic histopathological changes in SDT rats. Unexpectedly, CO dose-dependently exacerbated the impairment of kidney and mitochondrial functions caused by diabetes. CO also disturbed the expressions of proteins involved in mitochondrial biogenesis, dynamics, and mitophagy. However, no significant alterations were observed in nondiabetic rats receiving high-dose CO. The findings reveal that CO has deleterious effects that aggravate diabetic kidney injury via disrupting mitochondrial homeostasis, possibly due to its improper omega-6: omega-3 ratio.

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.

Endometriosis is a chronic disease characterized by the implantation and proliferation of endometrial tissue outside of the uterine cavity. The nonspecific nature of the symptoms and the lack of sensitive, noninvasive diagnostic methods often lead to a significant delay in diagnosis, highlighting the need for diagnostic biomarkers. The correlation of circulating miRNAs with altered inflammatory signals seen in patients with endometriosis has raised the possibility that miRNAs can serve as biomarkers for the disease. In our study, we analyzed miRNA expression in saliva of women with and without endometriosis using a FireFly custom multiplex circulating miRNA assay. This focused panel included 28 human miRNAs, 25 of which have been previously found to be differentially expressed either in plasma, serum, and/or blood of women with endometriosis, compared to controls. We found that hsa-mir-135a was expressed significantly higher in the saliva of women with endometriosis, independent of disease stage and menstrual cycle phase. We confirmed that hsa-mir-135a also showed significantly elevated expression in the plasma of endometriosis patients. This indicates that hsa-mir-135a is a putative noninvasive biomarker of endometriosis in both saliva and plasma, but further validation studies are required to assess its clinical value as a biomarker.

Multiple myeloma is characterized by a wide clinical heterogeneity due to an intricate network of interactions between bone marrow-resident clonal plasma cells and the microenvironment. Over the last years, dramatic improvement in the understanding of these pathways led to the introduction of novel drugs with immune-mediated mechanisms of action. Some of these compounds, such as the anti-cd38 daratumumab and isatuximab, the anti-slamf-7 elotuzumab, and the antibody-drug conjugate belantamab-mafodotin, have been tested in large clinical trials and have now fully entered the real-life management. The bispecific T-cell engagers are under investigation with promising results, and other satisfactory data is expected from the application of nanotechnologies. The perfect timing to introduce these drugs in the sequence of treatment and their adverse events represent new challenges to be addressed, and further experience is required to improve their use.

BACKGROUND: Epitranscriptomic mechanisms, such as A-to-I RNA editing mediated by ADAR deaminases, contribute to cancer heterogeneity and patients' stratification. ADAR enzymes can change the sequence, structure, and expression of several RNAs, affecting cancer cell behavior. In glioblastoma, an overall decrease in ADAR2 RNA level/activity has been reported. However, no data on ADAR2 protein levels in GBM patient tissues are available; and most data are based on ADARs overexpression experiments. METHODS: We performed IHC analysis on GBM tissues and correlated ADAR2 levels and patients' overall survival. We silenced ADAR2 in GBM cells, studied cell behavior, and performed a gene expression/editing analysis. RESULTS: GBM tissues do not all show a low/no ADAR2 level, as expected by previous studies. Although, different amounts of ADAR2 protein were observed in different patients, with a low level correlating with a poor patient outcome. Indeed, reducing the endogenous ADAR2 protein in GBM cells promotes cell proliferation and migration and changes the cell's program to an anchorage-independent growth mode. In addition, deep-seq data and bioinformatics analysis indicated multiple RNAs are differently expressed/edited upon siADAR2. CONCLUSION: ADAR2 protein is an important deaminase in GBM and its amount correlates with patient prognosis.

BACKGROUND: Cardiovascular events are the main cause of death in patients with chronic kidney disease. We hypothesize that the protective effects of renal cholesterol and vitamin D3 metabolism are lost under this condition. Nephropathy was induced by adenine in Apolipoprotein E knockout mice. The atherosclerotic phenotype was compared to mice with normal renal function. METHODS: Mice were fed a western diet ±0.15% adenine. Urine and feces were collected to assess renal function and fecal output. Atherosclerosis, serum lipoprotein composition and functionality, hepatic lipids, and expression of genes involved in lipid metabolism, vitamin D3 and Na+ homeostasis, were assessed. Bones were analyzed by microCT. RESULTS: Mice fed with adenine showed enhanced urinary Na+, Ca2+, and Pi excretion, reduced urinary pH, UreaUrine/UreaSerum, and CreatinineUrine/CreatinineSerum ratios. They developed less atherosclerosis. Lipoproteins in serum and hepatic lipids remained unchanged. Cholesterol efflux increased. Fecal output of cholesteryl ester and triglycerides increased. In the liver, mRNA levels of Cyp27a1, Cyp7a1, and Scarb1 increased; in the kidneys, Slc9a3, Slc12a3, Vdr, and Cyp24a1 decreased. Adenine increased cholesterol efflux in vitro. Tibias were shorter. CONCLUSION: Adenine induced tubular damage and was athero-protective because of enhanced cholesterol efflux and lipids elimination in feces. Bone growth was also affected.

Homomultimerization of MT1-MMP (membrane type 1 matrix metalloproteinase) through the hemopexin, transmembrane, and cytoplasmic domains plays a very important role in the activation of proMMP-2 and the degradation of pericellular collagen. MT1-MMP is overexpressed in many types of cancers, and it is considered to be a key enzyme in facilitating cancer cell migration. Since the oligomerization of MT1-MMP is important for its proteolytic activity in promoting cancer invasion, we have further investigated the multimerization by using heterologously expressed MT1-MMP ectodomains in insect cells to gain additional mechanistic insight into this process. We show that the whole ectodomain of MT1-MMP can form dimers and higher-order oligomeric complexes. The enzyme is secreted in its active form and the multimeric complex assembly is mediated by the catalytic domain. Blocking the prodomain removal determines the enzyme to adopt the monomeric structure, suggesting that the prodomain prevents the MT1-MMP oligomerization process. The binding affinity of MT1-MMP to type I collagen is dependent on the oligomeric state. Thus, the monomers have the weakest affinity, while the binding strength increases proportionally with the complexity of the multimers. Collectively, our experimental results indicate that the catalytic domain of MT1-MMP is necessary and sufficient to mediate the formation of multimeric structures.

The final maturation step of the 60S ribosomal subunit requires the release of eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) to enter the pool of mature ribosomes capable of engaging in translation. This process is mediated by the concerted action of the Elongation Factor-like 1 (human EFL1, yeast Efl1) GTPase and its effector, the Shwachman-Bodian-Diamond syndrome protein (human SBDS, yeast Sdo1). Mutations in these proteins prevent the release of eIF6 and cause a disease known as Shwachman-Diamond Syndrome (SDS). While some mutations in EFL1 or SBDS result in insufficient proteins to meet the cell production of mature large ribosomal subunits, others do not affect the expression levels with unclear molecular defects. We studied the functional consequences of one such mutation using Saccharomyces cerevisiae Efl1 R1086Q, equivalent to human EFL1 R1095Q described in SDS patients. We characterised the enzyme kinetics and energetic basis outlining the recognition of this mutant to guanine nucleotides and Sdo1, and their interplay in solution. From our data, we propose a model where the conformational change in Efl1 depends on a long-distance network of interactions that are disrupted in mutant R1086Q, whereby Sdo1 and the guanine nucleotides no longer elicit the conformational changes previously described in the wild-type protein. These findings point to the molecular malfunction of an EFL1 mutant and its possible impact on SDS pathology.

In humans, prenatal alcohol exposure can cause serious health issues in children, known collectively as Foetal Alcohol Spectrum Disorders (FASD). Despite the high prevalence of FASD and a lack of effective treatments, the underlying mechanisms causing the teratogenic action of ethanol are still obscure. The limitations of human studies necessitate the use of animal models for identifying the underlying processes, but few studies have investigated the effects of alcohol in the female germline. Here, we used the zebrafish Danio rerio to investigate the effects of chronic (repeated for seven days) exposure to alcohol. Specifically, we tested whether the offspring of females chronically exposed to ethanol during oogenesis exhibited hormonal abnormalities when subjected to a stressor (alarm cue) as larvae, and if they exhibited anxiety-like behaviours as adults. Exposure to alarm cue increased whole-body cortisol in control larvae but not in those of ethanol-treated females. Furthermore, adult offspring of ethanol-treated females showed some reduced anxiety-like behaviours. These findings suggest that the offspring of ethanol-treated females had reduced stress responses. This study is the first to investigate how maternal chronic ethanol exposure prior to fertilisation influences hormonal and behavioural effects in a non-rodent model.

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus.

Neurogenesis plays an important role in the prognosis of stroke patients and is known to be promoted by the activation of the Notch1 signaling pathway. Studies on the airway epithelium have shown that miR-449b represses the Notch pathway. The study aimed to investigate whether limb remote ischemic conditioning (LRIC) was able to promote neurogenesis in cerebral ischemic mice, and to investigate the role of the miR-449b/Notch1 pathway in LRIC-induced neuroprotection. Male C57BL/6 mice (22-25 g) were subjected to transient middle cerebral artery occlusion (MCAO), and LRIC was performed in the bilateral lower limbs immediately after MCA occlusion. Immunofluorescence staining was performed to assess neurogenesis. The cell line NE-4C was used to elucidate the proliferation of neuronal stem cells in 8% O2. After LRIC treatment on day 28, mice recovered neurological function. Neuronal precursor proliferation was enhanced in the SVZ, and neuronal precursor migration was enhanced in the basal ganglia on day 7. LRIC promoted the improvement of neurological function in mice on day 28, promoted neuronal precursor proliferation in the SVZ, and enhanced neuronal precursor migration in the basal ganglia on day 7. The neurological function score was negatively correlated with the number of BrdU-positive/DCX-positive cells in the SVZ and striatum. LRIC promoted activated Notch1 protein expression in the SVZ and substantially downregulated miR-449b levels in the SVZ and plasma. In vitro, miR-449b was found to target Notch1. Lentivirus-mediated miR-449b knockdown increased Notch1 levels in NE-4C cells and increased proliferation in the cells. The effects of miR-449b inhibition on neurogenesis were ablated by the application of Notch1 shRNA. Our study showed that LRIC promoted the proliferation and migration of neural stem cells after MCAO, and these effects were modulated by the miR-449b/Notch1 pathway.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

The UNited RESidue (UNRES) model of polypeptide chains was applied to study the association of 20 peptides with sizes ranging from 6 to 32 amino-acid residues. Twelve of those were potentially aggregating hexa- or heptapeptides excised from larger proteins, while the remaining eight contained potentially aggregating sequences, functionalized by attaching larger ends rich in charged residues. For 13 peptides, the experimental data of aggregation were used. The remaining seven were synthesized, and their properties were measured in this work. Multiplexed replica-exchange simulations of eight-chain systems were conducted at 12 temperatures from 260 to 370 K at concentrations from 0.421 to 5.78 mM, corresponding to the experimental conditions. The temperature profiles of the fractions of monomers and octamers showed a clear transition corresponding to aggregate dissociation. Low simulated transition temperatures were obtained for the peptides, which did not precipitate after incubation, as well as for the H-GNNQQNY-NH2 prion-protein fragment, which forms small fibrils. A substantial amount of inter-strand β-sheets was found in most of the systems. The results suggest that UNRES simulations can be used to assess peptide aggregation except for glutamine- and asparagine-rich peptides, for which a revision of the UNRES sidechain-sidechain interaction potentials appears necessary.

Pleiotropy, which refers to the ability of different mutations on the same gene to cause different pathological effects in human genetic diseases, is important in understanding system-level biological diseases. Although some biological experiments have been proposed, still little is known about pleiotropy on gene-gene dynamics, since most previous studies have been based on correlation analysis. Therefore, a new perspective is needed to investigate pleiotropy in terms of gene-gene dynamical characteristics. To quantify pleiotropy in terms of network dynamics, we propose a measure called in silico Pleiotropic Scores (sPS), which represents how much a gene is affected against a pair of different types of mutations on a Boolean network model. We found that our model can identify more candidate pleiotropic genes that are not known to be pleiotropic than the experimental database. In addition, we found that many types of functionally important genes tend to have higher sPS values than other genes; in other words, they are more pleiotropic. We investigated the relations of sPS with the structural properties in the signaling network and found that there are highly positive relations to degree, feedback loops, and centrality measures. This implies that the structural characteristics are principles to identify new pleiotropic genes. Finally, we found some biological evidence showing that sPS analysis is relevant to the real pleiotropic data and can be considered a novel candidate for pleiotropic gene research. Taken together, our results can be used to understand the dynamics pleiotropic characteristics in complex biological systems in terms of gene-phenotype relations.

The blood-brain barrier (BBB) is an essential structure for the maintenance of brain homeostasis. Alterations to the BBB are linked with a myriad of pathological conditions and play a significant role in the onset and evolution of neurodegenerative diseases, including Alzheimer's disease. Thus, a deeper understanding of the BBB's structure and function is mandatory for a better knowledge of neurodegenerative disorders and the development of effective therapies. Because studying the BBB in vivo imposes overwhelming difficulties, the in vitro approach remains the main possible way of research. With many in vitro BBB models having been developed over the last years, the main aim of this review is to systematically present the most relevant designs used in neurological research. In the first part of the article, the physiological and structural-functional parameters of the human BBB are detailed. Subsequently, available BBB models are presented in a comparative approach, highlighting their advantages and limitations. Finally, the new perspectives related to the study of Alzheimer's disease with the help of novel devices that mimic the in vivo human BBB milieu gives the paper significant originality.

Despite major advances in immunosuppression, allograft rejection remains an important complication after heart transplantation, and it is associated with increased morbidity and mortality. The gold standard invasive strategy to monitor and diagnose cardiac allograft rejection, based on the pathologic evaluation of endomyocardial biopsies, suffers from many limitations including the low prevalence of rejection, sample bias, high inter-observer variability, and international working formulations based on arbitrary cut-offs that simplify the landscape of rejection. The development of innovative diagnostic and prognostic strategies-integrating conventional histology, molecular profiling of allograft biopsy, and the discovery of new tissue or circulating biomarkers-is one of the major challenges of translational medicine in solid organ transplantation, and particularly in heart transplantation. Major advances in the field of biomarkers of rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. We review the recent developments in the field, including non-invasive biomarkers to minimize the number of protocol endomyocardial biopsies and tissue biomarkers as companion tools of pathology to refine the diagnosis of cardiac rejection. Finally, we discuss the potential role of these biomarkers to provide an integrated bio-histomolecular diagnosis of cardiac allograft rejection.

The detection of differentially expressed genes (DEGs) is one of most important computational challenges in the analysis of single-cell RNA sequencing (scRNA-seq) data. However, due to the high heterogeneity and dropout noise inherent in scRNAseq data, challenges in detecting DEGs exist when using a single distribution of gene expression levels, leaving much room to improve the precision and robustness of current DEG detection methods. Here, we propose the use of a new method, DEGman, which utilizes several possible diverse distributions in combination with Bhattacharyya distance. DEGman can automatically select the best-fitting distributions of gene expression levels, and then detect DEGs by permutation testing of Bhattacharyya distances of the selected distributions from two cell groups. Compared with several popular DEG analysis tools on both large-scale simulation data and real scRNA-seq data, DEGman shows an overall improvement in the balance of sensitivity and precision. We applied DEGman to scRNA-seq data of TRAP; Ai14 mouse neurons to detect fear-memory-related genes that are significantly differentially expressed in neurons with and without fear memory. DEGman detected well-known fear-memory-related genes and many novel candidates. Interestingly, we found 25 DEGs in common in five neuron clusters that are functionally enriched for synaptic vesicles, indicating that the coupled dynamics of synaptic vesicles across in neurons plays a critical role in remote memory formation. The proposed method leverages the advantage of the use of diverse distributions in DEG analysis, exhibiting better performance in analyzing composite scRNA-seq datasets in real applications.

Outcomes for patients following major peripheral nerve injury are extremely poor. Despite advanced microsurgical techniques, the recovery of function is limited by an inherently slow rate of axonal regeneration. In particular, a time-dependent deterioration in the ability of the distal stump to support axonal growth is a major determinant to the failure of reinnervation. Schwann cells (SC) are crucial in the orchestration of nerve regeneration; their plasticity permits the adoption of a repair phenotype following nerve injury. The repair SC modulates the initial immune response, directs myelin clearance, provides neurotrophic support and remodels the distal nerve. These functions are critical for regeneration; yet the repair phenotype is unstable in the setting of chronic denervation. This phenotypic instability accounts for the deteriorating regenerative support offered by the distal nerve stump. Over the past 10 years, our understanding of the cellular machinery behind this repair phenotype, in particular the role of c-Jun, has increased exponentially, creating opportunities for therapeutic intervention. This review will cover the activation of the repair phenotype in SC, the effects of chronic denervation on SC and current strategies to 'hack' these cellular pathways toward supporting more prolonged periods of neural regeneration.

Metastatic alveolar rhabdomyosarcoma (aRMS) is an aggressive paediatric cancer with a poor prognosis. Downregulation of critical tumour genes using targeted siRNA remains an obstacle, but association with nanoparticles could help to deliver, protect, target, and enhance penetration. siRNA towards two genes was investigated: (i) Human αB-crystallin (CRYAB) and Heat Shock Protein Family B (Small) Member 2 (HSPB2), and (ii) Keratin 17 (KRT17). A mesoporous silica based nanosystem was linked to siRNA via disulfide bonds and loaded with IR820 dye. Transfection efficiency and signalling was evaluated, and the metabolic effects and cell proliferation were monitored in 2D culture and 3D spheroid models. The bound siRNA was protected from degradation with RNase I for at least 24 h. The delivered siRNA showed significant suppression of viability; 53.21 ± 23.40% for CRYAB and HSPB2 siRNA, and 88.06 ± 17.28% for KRT17 siRNA. After 72 h this increased to >50% cell apoptosis and necrosis. Intracellular total glutathione (GSH) levels were also compared with fibroblasts, and the RMS cell lines showed a several-fold increase. IR820 cellular uptake rate and penetration depth was significantly improved by nanoparticle delivery. Targetted siRNA delivery may pave the way for less invasive and more effective treatments of aRMS.

Protein phase separation is increasingly understood to be an important mechanism of biological organization and biomaterial formation. Intrinsically disordered protein regions (IDRs) are often significant drivers of protein phase separation. A number of protein phase-separation-prediction algorithms are available, with many being specific for particular classes of proteins and others providing results that are not amenable to the interpretation of the contributing biophysical interactions. Here, we describe LLPhyScore, a new predictor of IDR-driven phase separation, based on a broad set of physical interactions or features. LLPhyScore uses sequence-based statistics from the RCSB PDB database of folded structures for these interactions, and is trained on a manually curated set of phase-separation-driving proteins with different negative training sets including the PDB and human proteome. Competitive training for a variety of physical chemical interactions shows the greatest contribution of solvent contacts, disorder, hydrogen bonds, pi-pi contacts, and kinked beta-structures to the score, with electrostatics, cation-pi contacts, and the absence of a helical secondary structure also contributing. LLPhyScore has strong phase-separation-prediction recall statistics and enables a breakdown of the contribution from each physical feature to a sequence's phase-separation propensity, while recognizing the interdependence of many of these features. The tool should be a valuable resource for guiding experiments and providing hypotheses for protein function in normal and pathological states, as well as for understanding how specificity emerges in defining individual biomolecular condensates.

The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m6A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m6A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m6A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

Pesticides are routinely used to prevent severe losses in agriculture. This practice is under debate because of its potential negative environmental impact and selection of resistances in pathogens. Therefore, the development of disease resistant plants is mandatory. It was shown that the rice (Oryza sativa) protein OsJAC1 enhances resistance against different bacterial and fungal plant pathogens in rice, barley, and wheat. Recently we reported possible carbohydrate interaction partners for both domains of OsJAC1 (a jacalin-related lectin (JRL) and a dirigent (DIR) domain), however, a mechanistic understanding of its function is still lacking. Here, we report crystal structures for both individual domains and the complex of galactobiose with the DIR domain, which revealed a new carbohydrate binding motif for DIR proteins. Docking studies of the two domains led to a model of the full-length protein. Our findings offer insights into structure and binding properties of OsJAC1 and its possible function in pathogen resistance.

The application of chicken waste to farmland could be detrimental to public health. It may contribute to the dissemination of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) from feces and their subsequent entry into the food chain. The present study analyzes the metagenome and resistome of chicken manure and litter obtained from a commercial chicken farm in Poland. ARB were isolated, identified, and screened for antibiogram fingerprints using standard microbiological and molecular methods. The physicochemical properties of the chicken waste were also determined. ARGs, integrons, and mobile genetic elements (MGE) in chicken waste were analyzed using high-throughput SmartChip qPCR. The results confirm the presence of many ARGs, probably located in MGE, which can be transferred to other bacteria. Potentially pathogenic or opportunistic microorganisms and phytopathogens were isolated. More than 50% of the isolated strains were classified as being multi-drug resistant, and the remainder were resistant to at least one antibiotic class; these pose a real risk of entering the groundwater and contaminating the surrounding environment. Our results indicate that while chicken manure can be sufficient sources of the nutrients essential for plant growth, its microbiological aspects make this material highly dangerous to the environment.

To provide precision medicine for better cancer care, researchers must work on clinical patient data, such as electronic medical records, physiological measurements, biochemistry, computerized tomography scans, digital pathology, and the genetic landscape of cancer tissue. To interpret big biodata in cancer genomics, an operational flow based on artificial intelligence (AI) models and medical management platforms with high-performance computing must be set up for precision cancer genomics in clinical practice. To work in the fast-evolving fields of patient care, clinical diagnostics, and therapeutic services, clinicians must understand the fundamentals of the AI tool approach. Therefore, the present article covers the following four themes: (i) computational prediction of pathogenic variants of cancer susceptibility genes; (ii) AI model for mutational analysis; (iii) single-cell genomics and computational biology; (iv) text mining for identifying gene targets in cancer; and (v) the NVIDIA graphics processing units, DRAGEN field programmable gate arrays systems and AI medical cloud platforms in clinical next-generation sequencing laboratories. Based on AI medical platforms and visualization, large amounts of clinical biodata can be rapidly copied and understood using an AI pipeline. The use of innovative AI technologies can deliver more accurate and rapid cancer therapy targets.

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular contraction are diminished in middle-aged animals. We determined the basal levels of HSP70 in the aorta of young and middle-aged Sprague Dawley male rats using Western blotting. Functional studies were performed in a wire myograph system. Force development in response to phenylephrine was assessed in the presence or absence of extracellular calcium (Ca2+), and in aortic rings treated or non-treated with an HSP70 inhibitor. Fluorescent probes were used to evaluate vascular oxidative stress and nitric oxide levels. We report that middle-aged rats have significantly lower levels of HSP70. Blockade of HSP70 attenuated vascular phasic and tonic contraction in isolated aortas. It appears that a functional HSP70 is required for proper Ca2+ handling as inhibition of this protein led to reduced force-displacement in response to Ca2+ dynamics. Furthermore, middle-aged aortic rings exposed to the HSP70 inhibitor display higher reactive oxygen species levels without changes in nitric oxide. In summary, we show that middle-aged animals have lower levels of HSP70 in aortas, which associates with an age-related decline in vascular responses to α-1 adrenergic stimulation.

Angiosarcoma is a rare, devastating malignancy with few curative options for disseminated disease. We analyzed a recently published genomic data set of 48 angiosarcomas and noticed recurrent amplifications of HOXA-cluster genes in 33% of patients. HOXA genes are master regulators of embryonic vascular development and adult neovascularization, which provides a molecular rationale to suspect that amplified HOXA genes act as oncogenes in angiosarcoma. HOXA amplifications typically affected multiple pro-angiogenic HOXA genes and co-occurred with amplifications of CD36 and KDR, whereas the overall mutation rate in these tumors was relatively low. HOXA amplifications were found most commonly in angiosarcomas located in the breast and were rare in angiosarcomas arising in sun-exposed areas on the head, neck, face and scalp. Our data suggest that HOXA-amplified angiosarcoma is a distinct molecular subgroup. Efforts to develop therapies targeting oncogenic HOX gene expression in AML and other sarcomas may have relevance for HOXA-amplified angiosarcoma.

The reprogrammable CRISPR/Cas9 genome editing tool's growing popularity is hindered by unwanted off-target effects. Efforts have been directed toward designing efficient guide RNAs as well as identifying potential off-target threats, yet factors that determine efficiency and off-target activity remain obscure. Based on sequence features, previous machine learning models performed poorly on new datasets, thus there is a need for the incorporation of novel features. The binding energy estimation of the gRNA-DNA hybrid as well as the Cas9-gRNA-DNA hybrid allowed generating better performing machine learning models for the prediction of Cas9 activity. The analysis of feature contribution towards the model output on a limited dataset indicated that energy features played a determining role along with the sequence features. The binding energy features proved essential for the prediction of on-target activity and off-target sites. The plateau, in the performance on unseen datasets, of current machine learning models could be overcome by incorporating novel features, such as binding energy, among others. The models are provided on GitHub (GitHub Inc., San Francisco, CA, USA).

The structure of the transmembrane domain of the pH-activated bacterial potassium channel KcsA has been extensively characterized, yet little information is available on the structure of its cytosolic, functionally critical N- and C-termini. This study presents high-resolution magic angle spinning (HR-MAS) and fractional deuteration as tools to study these poorly resolved regions for proteoliposome-embedded KcsA. Using 1H-detected HR-MAS NMR, we show that the C-terminus transitions from a rigid structure to a more dynamic structure as the solution is rendered acidic. We make previously unreported assignments of residues in the C-terminus of lipid-embedded channels. These data agree with functional models of the C-terminus-stabilizing KcsA tetramers at a neutral pH with decreased stabilization effects at acidic pH. We present evidence that a C-terminal truncation mutation has a destabilizing effect on the KcsA selectivity filter. Finally, we show evidence of hydrolysis of lipids in proteoliposome samples during typical experimental timeframes.

This study compared the physicochemical and functional properties of starches from eight cultivars of avocado seeds. Amylose content, morphology, crystalline structure, swelling power, solubility, thermal and pasting properties, and in vitro digestibility were investigated. The results revealed that the apparent amylose content of starches from avocado seeds varied with different varieties. Light microscopic and scanning electron microscopic examination demonstrated that the eight starches differed slightly in terms of morphology and granule size. The X-ray diffraction and Fourier transform infrared spectroscopy analyses showed that the crystal structure and chemical linkage of the avocado seed starches were similar. However, the pasting, water solubility, and thermal properties of the eight avocado seed starches differed. Importantly, all the starches had high resistant starch content (>60%), with the highest found in Hass seeds (77.83%). To conclude, starch from avocado seeds has a high potential for use in the production of resistant starch.

Phenylacetylglutamine (PAGln), a gut microbiota (GM)-derived metabolite, is associated with cardiovascular disease. Studies have shown that disordered GM participated in the progression of atrial fibrillation (AF), but the relationship between PAGln and AF is unclear. This study investigated the characteristics of PAGln in AF patients and its impact on atrial myocytes. Based on our previous metagenomic data, the relative abundance of porA, a critical bacterial enzyme for PAGln synthesis, exhibited an increased tendency in AF. In an independent cohort consisting of 42 controls without AF and 92 AF patients, plasma PAGln levels were higher in AF patients than in controls (p < 0.001) by immunoassay. Notably, PAGln exerted a predictive potential of AF with an AUC of 0.774 (p < 0.001), and a predictive model constructed based on the PAGln and Taiwan AF score further improved the predictive potential. Furthermore, a positive correlation was determined between PAGln and LA diameter. Subsequently, the effect of PAGln intervention was examined on HL-1 cells in vitro, revealing that PAGln increased apoptosis, reactive oxygen species (ROS) production, CaMKII and RyR2 activation and decreased cell viability. In conclusion, increased PAGln was associated with AF, and PAGln might contribute to the AF pathogenesis by promoting oxidative stress and apoptosis in atrial myocytes.

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor Gs signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the Gq/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on Gq/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRETTM studies to determine protein-protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. Gq/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on Gq/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity.

Several studies suggest that different combinations of nutraceutical supplements may improve the lipid profile, representing a viable alternative to statins. However, their effects on individuals with myopathy need to be investigated. The aim of our study was to explore the mid- and long-term physiological effects of monacolin k (5 mg) and astaxanthin (0.1 mg) supplements in association with a low-energy/fat diet in a group of subjects with mild myopathy. Eighty subjects (44 women) took part in this observational study. Participants were assigned to the experimental group (EG, n = 40, 24 women) treated with a low-energy/fat diet (1200-1500 Kcal/day and 15-20% lipids) in combination with monacolin k (5 mg) and astaxanthin (0.1 mg) supplementation, and to the control group (CG, n = 40, 20 women) treated only with a low-energy/fat diet (1200-1500 Kcal/day and 15-20% lipids). BMI and biochemical parameters (blood glucose, total cholesterol, HDL, LDL, triglycerides, C-reactive protein (CRP) and creatine phosphokinase-CPK) were collected at baseline (T0), after 12 (T1) and 24 (T2) weeks. A mixed factorial ANOVA was performed to determine if there were significant main effects and/or interactions between time and treatment. Treatment (EG vs. CG) was entered as the between-subjects factor and time (T0 vs. T1 vs. T2) as the within-subject factor. We found a significant improvement in total cholesterol, HDL, LDL, PCR and CPK parameters in EG compared with CG. Our results highlight the efficacy and safety of combined use of monacolin k (5 mg) and astaxanthin (0.1 mg) in combination with a low-energy/fat diet in the treatment of dyslipidemia.

BACKGROUND: The aim of the study was to define whether edaravone, a free-radical scavenger, influenced angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysms (AAAs) formation. METHODS: Male apolipoprotein E-deficient mice (8-12 weeks old) were fed with a normal diet for 5 weeks. Either edaravone (10 mg/kg/day) or vehicle was injected intraperitoneally for 5 weeks. After 1 week of injections, mice were infused subcutaneously with either AngII (1000 ng/kg/min, n = 16-17 per group) or saline (n = 5 per group) by osmotic minipumps for 4 weeks. RESULTS: AngII increased systolic blood pressure equivalently in mice administered with either edaravone or saline. Edaravone had no effect on plasma total cholesterol concentrations and body weights. AngII infusion significantly increased ex vivo maximal diameters of abdominal aortas and en face atherosclerosis but was significantly attenuated by edaravone administration. Edaravone also reduced the incidence of AngII-induced AAAs. In addition, edaravone diminished AngII-induced aortic MMP-2 activation. Quantitative RT-PCR revealed that edaravone ameliorated mRNA abundance of aortic MCP-1 and IL-1β. Immunostaining demonstrated that edaravone attenuated oxidative stress and macrophage accumulation in the aorta. Furthermore, edaravone administration suppressed thioglycolate-induced mice peritoneal macrophages (MPMs) accumulation and mRNA abundance of MCP-1 in MPMs in male apolipoprotein E-deficient mice. In vitro, edaravone reduced LPS-induced mRNA abundance of MCP-1 in MPMs. CONCLUSIONS: Edaravone attenuated AngII-induced AAAs and atherosclerosis in male apolipoprotein E-deficient mice via anti-oxidative action and anti-inflammatory effect.

BACKGROUND: Due to resistance to conventional therapy, a blood-brain barrier that results in poor drug delivery, and a high potential for metastasis, glioblastoma (GBM) presents a great medical challenge. Since the repertoire of the possible therapies is very limited, novel therapeutic strategies require new drugs as well as new approaches. The multiple roles played by L-tryptophan (Trp) in tumorigenesis of GBM and the previously found antiproliferative properties of Trp-bearing dendrimers against this malignancy prompted us to design novel polyfunctional peptide-based dendrimers covalently attached to N1-alkyl tryptophan (Trp) residues. Their antiproliferative properties against GBM and normal human astrocytes (NHA) and their antioxidant potential were tested. METHODS: Two groups of amphiphilic peptide dendrimers terminated with N1-butyl and N1-aminopentane tryptophan were designed. The influence of dendrimers on viability of NHA and human GBM cell lines, displaying different genetic backgrounds and tumorigenic potentials, was determined by the MTT test. The influence of compounds on the clonogenic potential of GBM cells was assessed by colony-formation assay. Dendrimers were tested for radical scavenging potency as well as redox capability (DPPH, ABTS, and FRAP models). RESULTS: Several peptide dendrimers functionalized with N1-alkyl-tryptophan at 5 µM concentration exhibited high selectivity towards GBM cells retaining 85-95% viable NHA cells while killing cancer cells. In both the MTT and colony-formation assays, compounds 21 (functionalized with N1-butyl-Trp and (+)8 charged) and 25 (functionalized with N1-aminopentane-Trp and (+)12 charged) showed the most promise for their development into anticancer drugs. According to ABTS, DPPH, and FRAP antioxidant tests, dendrimers functionalized with N1-alkylated Trp expressed higher ROS-scavenging capacity (ABTS and DPPH) than those with unsubstituted Trp. CONCLUSIONS: Peptide dendrimers functionalized with N1-alkyl-tryptophan showed varying toxicity to NHA, while all were toxic to GBM cells. Based on their activity towards inhibition of GBM viability and relatively mild effect on NHA cells the most advantageous were derivatives 21 and 25 with the respective di-dodecyl and dodecyl residue located at the C-terminus. As expected, peptide dendrimers functionalized with N1-alkyl-tryptophan expressed higher scavenging potency against ROS than dendrimers with unsubstituted tryptophan.

Centrins are calcium (Ca2+)-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from Toxoplasma gondii (TgCEN1). However, the molecular details of the interaction between TgCEN1 and TgSFI1 remain largely unknown. Herein, combining different biophysical methods, including isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, we determined the binding properties of TgCEN1 and its individual N- and C-terminal domains to synthetic peptides derived from distinct repeats of TgSFI1. Overall, our data indicate that the repeats in TgSFI1 constitute binding sites for TgCEN1, but the binding modes of TgCEN1 to the repeats differ appreciably in terms of binding affinity, Ca2+ sensitivity, and lobe-specific interaction. These results suggest that TgCEN1 displays remarkable conformational plasticity, allowing for the distinct repeats in TgSFI1 to possess precise modes of TgCEN1 binding and regulation during Ca2+ sensing, which appears to be crucial for the dynamic association of TgCEN1 with TgSFI1 in the centrosome architecture.

Most neurodegenerative diseases are multifactorial, and the discovery of several molecular mechanisms related to their pathogenesis is constantly advancing. Dopamine and dopaminergic receptor subtypes are involved in the pathophysiology of several neurological disorders, such as schizophrenia, depression and drug addiction. For this reason, the dopaminergic system and dopamine receptor ligands play a key role in the treatment of such disorders. In this context, a novel series of conformationally restricted N-arylpiperazine derivatives (5a-f) with a good affinity for D2/D3 dopamine receptors is reported herein. Compounds were designed as interphenylene analogs of the drugs aripiprazole (2) and cariprazine (3), presenting a 1,3-benzodioxolyl subunit as a ligand of the secondary binding site of these receptors. The six new N-arylpiperazine compounds were synthesized in good yields by using classical methodologies, and binding and guanosine triphosphate (GTP)-shift studies were performed. Affinity values below 1 μM for both target receptors and distinct profiles of intrinsic efficacy were found. Docking studies revealed that Compounds 5a-f present a different binding mode with dopamine D2 and D3 receptors, mainly as a consequence of the conformational restriction imposed on the flexible spacer groups of 2 and 3.

Inhibition of Plasmodium falciparum nicotinamidase could represent a potential antimalarial since parasites require nicotinic acid to successfully recycle nicotinamide to NAD+, and importantly, humans lack this biosynthetic enzyme. Recently, mechanism-based inhibitors of nicotinamidase have been discovered. The most potent compound inhibits both recombinant P. falciparum nicotinamidase and parasites replication in infected human red blood cells (RBCs). These studies provide evidence for the importance of nicotinamide salvage through nicotinamidase as a central master player of NAD+ homeostasis in P. falciparum.

Diabetes mellitus is a common disease affecting more than 537 million adults worldwide. The microvascular complications that occur during the course of the disease are widespread and affect a variety of organ systems in the body. Diabetic retinopathy is one of the most common long-term complications, which include, amongst others, endothelial dysfunction, and thus, alterations in the blood-retinal barrier (BRB). This particularly restrictive physiological barrier is important for maintaining the neuroretina as a privileged site in the body by controlling the inflow and outflow of fluid, nutrients, metabolic end products, ions, and proteins. In addition, people with diabetic retinopathy (DR) have been shown to be at increased risk for systemic vascular complications, including subclinical and clinical stroke, coronary heart disease, heart failure, and nephropathy. DR is, therefore, considered an independent predictor of heart failure. In the present review, the effects of diabetes on the retina, heart, and kidneys are described. In addition, a putative common microRNA signature in diabetic retinopathy, nephropathy, and heart failure is discussed, which may be used in the future as a biomarker to better monitor disease progression. Finally, the use of miRNA, targeted neurotrophin delivery, and nanoparticles as novel therapeutic strategies is highlighted.

The purpose of this work is to develop a novel ultrathin fibrous membrane with a core-sheath structure as antibacterial food packaging film. Coaxial electrospinning was exploited to create the core-sheath structure, by which the delivery regulation of the active substance was achieved. Resveratrol (RE) and silver nanoparticles (AgNPs) were loaded into electrospun zein/polyethylene oxide ultrathin fibers to ensure a synergistic antibacterial performance. Under the assessments of a scanning electron microscope and transmission electron microscope, the ultrathin fiber was demonstrated to have a fine linear morphology, smooth surface and obvious core-sheath structure. X-ray diffraction and Fourier transform infrared analyses showed that RE and AgNPs coexisted in the ultrathin fibers and had good compatibility with the polymeric matrices. The water contact angle experiments were conducted to evaluate the hydrophilicity and hygroscopicity of the fibers. In vitro dissolution tests revealed that RE was released in a sustained manner. In the antibacterial experiments against Staphylococcus aureus and Escherichia coli, the diameters of the inhibition zone of the fiber were 8.89 ± 0.09 mm and 7.26 ± 0.10 mm, respectively. Finally, cherry tomatoes were selected as the packaging object and packed with fiber films. In a practical application, the fiber films effectively reduced the bacteria and decreased the quality loss of cherry tomatoes, thereby prolonging the fresh-keeping period of cherry tomatoes to 12 days. Following the protocols reported here, many new food packaging films can be similarly developed in the future.

Alpha synuclein (aSyn) and its aggregation are crucial for the neurodegeneration of Parkinson's disease (PD). aSyn was initially described in the nucleus and presynaptic nerve terminals. However, the biology of nuclear aSyn and the link of aSyn between subcellular compartments are less understood. Current knowledge suggests the existence of various aSyn species with distinct structural and biochemical properties. Here, we identified a C-terminal-targeting aSyn antibody (Nu-aSyn-C), which has a high immunoaffinity towards aSyn in the nucleus. Comparing the Nu-aSyn-C antibody to aSyn antibodies developed against phosphorylated or aggregated forms, we observed that nuclear aSyn differs from cytosolic aSyn by an increased phosphorylation and assembly level in proliferating cells. Employing Nu-aSyn-C, we characterized aSyn distribution during neuronal differentiation in midbrain dopaminergic neurons (mDANs) derived from human-induced pluripotent stem cells (hiPSCs) and Lund human mesencephalic cells, and in primary rat hippocampal neurons. We detected a specific translocation pattern of aSyn during neuronal differentiation from the nucleus to the soma and finally to neuronal processes. Interestingly, a remarkable shift of Nu-aSyn-C-positive species towards neurites was detected in hiPSC mDANs from a PD patient carrying aSyn gene duplication. Together, our results reveal distinct nuclear and cytosolic aSyn species that redistribute during neuronal differentiation-a process that is altered in PD-derived neurons.

Affibodies targeting intracellular proteins have a great potential to function as ideal therapeutic agents. However, little is known about how the affibodies enter target cells to interact with intracellular target proteins. We have previously developed the HPV16E7 affibody (ZHPV16E7384) for HPV16 positive cervical cancer treatment. Here, we explored the underlying mechanisms of ZHPV16E7384 and found that ZHPV16E7384 significantly inhibited the proliferation of target cells and induced a G1/S phase cell cycle arrest. Furthermore, ZHPV16E7384 treatment resulted in the upregulation of retinoblastoma protein (Rb) and downregulation of phosphorylated Rb (pRb), E2F1, cyclin D1, and CDK4 in the target cells. Moreover, treatment with dynamin or the caveolin-1 inhibitor not only significantly suppressed the internalization of ZHPV16E7384 into target cells but also reversed the regulation of cell cycle factors by ZHPV16E7384. Overall, these results indicate that ZHPV16E7384 was likely internalized specifically into target cells through dynamin- and caveolin-1 mediated endocytosis. ZHPV16E7384 induced the cell cycle arrest in the G1/S phase at least partially by interrupting HPV16E7 binding to and degrading Rb, subsequently leading to the downregulation of E2F1, cyclin D1, CDK4, and pRb, which ultimately inhibited target cell proliferation. These findings provide a rationale of using ZHPV16E7384 to conduct a clinical trial for target therapy in cervical cancer.

Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart's molecular characteristics.

Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. Here, we investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. Our findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA-HSA (Ka ≈ 103 L/mol) and moderately strong CPA-HSA and STC-HSA complexes (Ka ≈ 104 L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow's site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA-HSA interaction may have toxicological importance.

Osteogenesis imperfecta(OI) is a disease caused by substitution in glycine residues with different amino acids in type I collagen (Gly-Xaa-Yaa)n. Collagen model peptides can capture the thermal stability loss of the helix after Gly mutations, most of which are homotrimers. However, a majority of natural collagen exists in heterotrimers. To investigate the effects of chain specific mutations in the natural state of collagen more accurately, here we introduce various lengths of side-chain amino acids into ABC-type heterotrimers. The disruptive effects of the mutations were characterized both experimentally and computationally. We found the stability decrease in the mutants was mainly caused by the disruption of backbone hydrogen bonds. Meanwhile, we found a threshold value of local hydrogen bonding energy that could predict triple helix folding or unfolding. Val caused the unfolding of triple helices, whereas Ser with a similar side-chain length did not. Structural details suggested that the side-chain hydroxyl group in Ser forms hydrogen bonds with the backbone, thereby compensating for the mutants' decreased stability. Our study contributes to a better understanding of how OI mutations destabilize collagen triple helices and the molecular mechanisms underlying OI.

Sesquiterpene compounds are applied as permeation promoters in topical formulations. However, studies exploring their impact on nanostructured systems, changes in permeation profile, and consequently, its biological activity are restricted. This study aimed to investigate the correlation between the skin permeation of the major sesquiterpenes, beta-caryophyllene, and caryophyllene oxide from the oleoresin of Copaifera multijuga, after delivery into topical nanoemulgels, and the in vivo antiedematogenic activity. First, ten nanoemulgels were prepared and characterized, and their in vitro permeation profile and in vivo anti-inflammatory activity were evaluated. In equivalent concentrations, β-caryophyllene permeation was greater from oleoresin nanoemulgels, resulting in greater in vivo antiedematogenic activity. However, an inverse relationship was observed for caryophyllene oxide, which showed its favored permeation and better in vivo anti-inflammatory effect carried as an isolated compound in the nanoemulgels. These results suggest that the presence of similar compounds may interfere with the permeation profile when comparing the profiles of the compounds alone or when presented in oleoresin. Furthermore, the correlation results between the permeation profile and in vivo antiedematogenic activity corroborate the establishment of beta-caryophyllene as an essential compound for this pharmacological activity of C. multijuga oleoresin.

Irisin is an adipo-myokine, mainly synthetized in skeletal muscles and adipose tissues, that is involved in multiple processes. Only a few studies have evaluated serum irisin in psoriatic patients. This study aims to analyze serum irisin levels in patients with chronic plaque psoriasis, to compare them with values in controls, and to assess whether concentration of circulating irisin correlates with the severity of psoriasis, calculated by means of Psoriasis Area and Severity Index (PASI). We enrolled 46 patients with chronic plaque psoriasis; the control group included 46 sex- and age-matched subjects without any skin or systemic diseases. Serum irisin levels were measured by competitive enzyme linked immunosorbent assay. Our results showed a non-significant increase in serum irisin concentration in psoriatic patients compared to controls. A negative non-linear correlation between PASI and irisin levels was detected in psoriatic patients. Indeed, dividing patients according to psoriasis severity, the negative association between irisin and PASI was stronger in patients with mild psoriasis than in patients with higher PASI scores. Several control variables we tested showed no significant impact on serum irisin. However, erythrocyte sedimentation rate in the normal range was associated with significantly higher irisin levels in psoriatic patients. In conclusion, although irisin levels were not significantly different between controls and psoriatic patients, irisin was found to be negatively associated with psoriasis severity, especially in subjects with low PASI scores; however, further studies are needed to clarify the role of irisin in subjects with psoriasis.

Despite the variable chemical and physical characteristics of particulate air pollutants, inflammation and oxidative stress have been identified as common mechanisms for cell damage and negative health influences. These effects are produced by organic components, especially by endotoxins. This study analyzed the gene expression profile after exposure of RAW 264.7 cells to the standard particulate matter (PM) material, NIST1648a, and PM with a reduced organic matter content, LAp120, in comparison to the effects of lipopolysaccharide (LPS). The selected parameters of cell viability, cell cycle progression, and metabolic and inflammatory activity were also investigated. Both forms of PM negatively influenced the parameters of cell activity. These results were generally reflected in the gene expression profile. Only NIST1648a, excluding LAp120, contained endotoxins and showed small but statistically significant pro-inflammatory activity. However, the gene expression profiling revealed strong pro-inflammatory cell activation induced by NIST1648a that was close to the effects of LPS. Changes in gene expression triggered by LAp120 were relatively small. The observed differences in the effects of NIST1648a and LAp120 were related to the content of organic matter in which bacterial endotoxins play an important role. However, other organic compounds and their interactions with other PM components also appear to be of significant importance.

Blood phospho-tau181 may offer a useful biomarker for Alzheimer's disease. However, the use of either serum or plasma phospho-tau181 and their diagnostic value are currently under intense investigation. In a pilot study, we measured both serum and plasma phospho-tau181 (pT181-Tau) by single molecule array (Simoa) in a group of patients with Alzheimer's disease and a mixed group of patients with other primary dementing and/or movement disorders. Classical cerebrospinal fluid biomarkers were also measured. Plasma (but not serum) pT181-Tau showed a significant increase in Alzheimer's disease and correlated significantly with cerebrospinal fluid amyloid and pT181-Tau. Receiver operating curve analysis revealed a significant discrimination of Alzheimer's from non-Alzheimer's disease patients, with an area under the curve of 0.83 and an excellent sensitivity but a moderate specificity. Plasma pT181-Tau is not an established diagnostic biomarker for Alzheimer's disease, but it could become one in the future, or it may serve as a screening tool for specific cases of patients or presymptomatic subjects.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

There is increasing evidence that many intrinsically disordered regions (IDRs) in proteins play key functional roles through interactions with other proteins or nucleic acids. These interactions often exhibit a context-dependent structural behavior. We hypothesize that low complexity regions (LCRs), often found within IDRs, could have a role in inducing local structure in IDRs. To test this, we predicted IDRs in the human proteome and analyzed their structures or those of homologous sequences in the Protein Data Bank (PDB). We then identified two types of simple LCRs within IDRs: regions with only one (polyX or homorepeats) or with only two types of amino acids (polyXY). We were able to assign structural information from the PDB more often to these LCRs than to the surrounding IDRs (polyX 61.8% > polyXY 50.5% > IDRs 39.7%). The most frequently observed polyX and polyXY within IDRs contained E (Glu) or G (Gly). Structural analyses of these sequences and of homologs indicate that polyEK regions induce helical conformations, while the other most frequent LCRs induce coil structures. Our work proposes bioinformatics methods to help in the study of the structural behavior of IDRs and provides a solid basis suggesting a structuring role of LCRs within them.

Hirschsprung disease (HSCR) is characterised by the absence of enteric ganglia along variable lengths of the distal bowel. Current gold standard treatment involves the surgical resection of the defective, aganglionic bowel. Clear and reliable distinction of the normoganglionated bowel from the transition zone is key for successful resection of the entire defective bowel, and the avoidance of subsequent postoperative complications. However, the intraoperative nature of the tissue analysis and the variability of patient samples, sample preparation, and operator objectivity, make reproducible identification of the transition zone difficult. Here, we have described a novel method for using muscle units as a distinctive landmark for quantifying the density of enteric ganglia in resection specimens from HSCR patients. We show that the muscle unit to ganglion ratio is greater in the transition zone when compared with the proximal, normoganglionated region for long-segment HSCR patients. Patients with short-segment HSCR were also investigated, however, the muscle unit to ganglion ratio was not significantly different in these patients. Immunohistochemical examination of individual ganglia showed that there were no differences in the proportions of either enteric neurons or glial cells through the different regions of the resected colon. In addition, we identified that the size of enteric ganglia was smaller for patients that went on to develop HSCR associated enterocolitis; although the density of ganglia, as determined by the muscle unit to ganglia ratio, was not different when compared with patients that had no further complications. This suggests that subtle changes in the enteric nervous system, even in the "normoganglionated" colon, could be involved in changes in immune function and subsequent bacterial dysbiosis.

Creep-associated changes in disc bulging and axial strains are essential for the research and development of mechano-bionic biomaterials and have been assessed in various ways in ex vivo creep studies. Nonetheless, the reported methods for measurement were limited by location inaccuracy, a lack of synchronousness, and destructiveness. To this end, this study focuses on the accurate, synchronous, and noninvasive assessment of bugling and strains using the 3D digital image correlation (3D-DIC) system and the impact of creep on them. After a preload of 30 min, the porcine cervical discs were loaded with different loads for 4 h of creep. Axial strains and lateral bulging of three locations on the discs were synchronously measured. The three-parameter solid model and the newly proposed horizontal asymptote model were used to fit the acquired data. The results showed that the load application reduced disc strains by 6.39% under 300 N, 11.28% under 400 N, and 12.59% under 500 N. Meanwhile, the largest protrusion occurred in the middle of discs with a bugling of 1.50 mm, 1.67 mm, and 1.87 mm. Comparison of the peer results showed that the 3D-DIC system could be used in ex vivo biomechanical studies with reliability and had potential in the assessment of the mechanical behavior of novel biomaterials. The phenomenon of the largest middle protrusion enlightened further the strength of spinal implants in this area. The mathematical characterizations of bulging and strains under different loads yielded various model parameters, which are prerequisites for developing implanted biomaterials.

Coenzyme A (CoA) is ubiquitous and essential for key cellular processes in any living organism. Primary degradation of CoA occurs by enzyme-mediated pyrophosphate hydrolysis intracellularly and extracellularly to form adenosine 3',5'-diphosphate and 4'-phosphopantetheine (PPanSH). The latter can be recycled for intracellular synthesis of CoA. Impairments in the CoA biosynthetic pathway are linked to a severe form of neurodegeneration with brain iron accumulation for which no disease-modifying therapy is available. Currently, exogenous administration of PPanSH is examined as a therapeutic intervention. Here, we describe biosynthetic access to thiophosphate analogs of PPanSH, 3'-dephospho-CoA, and CoA. The stabilizing effect of thiophosphate modifications toward degradation by extracellular and peroxisomal enzymes was studied in vitro. Experiments in a CoA-deficient cell model suggest a biomimetic potential of the PPanSH thiophosphate analog PSPanSH (C1). According to our findings, the administration of PSPanSH may provide an alternative approach to support intracellular CoA-dependent pathways.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.