Obesogens, as environmental endocrine-disrupting chemicals, are supposed to have had an impact on the prevalence of rising obesity around the world over the last forty years. These chemicals are probably able to contribute not only to the development of obesity and metabolic disturbances in individuals, but also in their progeny, having the capability to epigenetically reprogram genetically inherited set-up points for body weight and body composition control during critical periods of development, such as fetal, early life, and puberty. In individuals, they may act on myriads of neuro-endocrine-immune metabolic regulatory pathways, leading to pathophysiological consequences in adipogenesis, lipogenesis, lipolysis, immunity, the influencing of central appetite and energy expenditure regulations, changes in gut microbiota-intestine functioning, and many other processes. Evidence-based medical data have recently brought much more convincing data about associations of particular chemicals and the probability of the raised risk of developing obesity. Foods are the main source of obesogens. Some obesogens occur naturally in food, but most are environmental chemicals, entering food as a foreign substance, whether in the form of contaminants or additives, and they are used in a large amount in highly processed food. This review article contributes to a better overview of obesogens, their occurrence in foods, and their impact on the human organism.

ABCA1 and ABCG1 are two ABC-transporters well-recognized to promote the efflux of cholesterol to apoAI and HDL, respectively. As these two ABC-transporters are critical to cholesterol metabolism, several studies have assessed the impact of ABCA1 and ABCG1 expression on cellular cholesterol homeostasis through ABC-transporter ablation or overexpressing ABCA1/ABCG1. However, for the latter, there are currently no well-established in vitro models to effectively induce long-term ABC-transporter expression in a variety of cultured cells. Therefore, we performed proof-of-principle in vitro studies to determine whether a LoxP-Stop-LoxP (LSL) system would provide Cre-inducible ABC-transporter expression. In our studies, we transfected HEK293 cells and the HEK293-derived cell line 293-Cre cells with ABCA1-LSL and ABCG1-LSL-based plasmids. Our results showed that while the ABCA1/ABCG1 protein expression was absent in the transfected HEK293 cells, the ABCA1 and ABCG1 protein expression was detected in the 293-Cre cells transfected with ABCA1-LSL and ABCG1-LSL, respectively. When we measured cholesterol efflux in transfected 293-Cre cells, we observed an enhanced apoAI-mediated cholesterol efflux in 293-Cre cells overexpressing ABCA1, and an HDL2-mediated cholesterol efflux in 293-Cre cells constitutively expressing ABCG1. We also observed an appreciable increase in HDL3-mediated cholesterol efflux in ABCA1-overexpressing 293-Cre cells, which suggests that ABCA1 is capable of effluxing cholesterol to small HDL particles. Our proof-of-concept experiments demonstrate that the LSL-system can be used to effectively regulate ABC-transporter expression in vitro, which, in turn, allows ABCA1/ABCG1-overexpression to be extensively studied at the cellular level.

Plants encounter several abiotic stresses, among which heat stress is gaining paramount attention because of the changing climatic conditions. Severe heat stress conspicuously reduces crop productivity through changes in metabolic processes and in growth and development. Ethylene and hydrogen sulfide (H2S) are signaling molecules involved in defense against heat stress through modulation of biomolecule synthesis, the antioxidant system, and post-translational modifications. Other compounds containing the essential mineral nutrient sulfur (S) also play pivotal roles in these defense mechanisms. As biosynthesis of ethylene and H2S is connected to the S-assimilation pathway, it is logical to consider the existence of a functional interplay between ethylene, H2S, and S in relation to heat stress tolerance. The present review focuses on the crosstalk between ethylene, H2S, and S to highlight their joint involvement in heat stress tolerance.

BACKGROUND: Alcohol-related brain degeneration is linked to cognitive-motor deficits and impaired signaling through insulin/insulin-like growth factor type 1 (IGF-1)-Akt pathways that regulate cell survival, plasticity, metabolism, and homeostasis. In addition, ethanol inhibits Aspartyl-asparaginyl-β-hydroxylase (ASPH), a downstream target of insulin/IGF-1-Akt signaling and an activator of Notch networks. Previous studies have suggested that early treatment with insulin sensitizers or dietary soy could reduce or prevent the long-term adverse effects of chronic ethanol feeding. OBJECTIVE: The goal of this study was to assess the effects of substituting soy isolate for casein to prevent or reduce ethanol's adverse effects on brain structure and function. METHODS: Young adolescent male and female Long Evans were used in a 4-way model as follows: Control + Casein; Ethanol + Casein; Control + Soy; Ethanol + Soy; Control = 0% ethanol; Ethanol = 26% ethanol (caloric). Rats were fed isocaloric diets from 4 to 11 weeks of age. During the final experimental week, the Morris Water maze test was used to assess spatial learning (4 consecutive days), after which the brains were harvested to measure the temporal lobe expression of the total phospho-Akt pathway and downstream target proteins using multiplex bead-based enzyme-linked immunosorbent assays (ELISAs) and duplex ELISAs. RESULTS: Ethanol inhibited spatial learning and reduced brain weight, insulin signaling through Akt, and the expression of ASPH when standard casein was provided as the protein source. The substitution of soy isolate for casein largely abrogated the adverse effects of chronic ethanol feeding. In contrast, Notch signaling protein expression was minimally altered by ethanol or soy isolate. CONCLUSIONS: These novel findings suggest that the insulin sensitizer properties of soy isolate may prevent some of the adverse effects that chronic ethanol exposure has on neurobehavioral function and insulin-regulated metabolic pathways in adolescent brains.

The kappa index (K-Index), calculated by dividing the cerebrospinal fluid (CSF)/serum kappa free light chain (KFLC) ratio by the CSF/serum albumin ratio, is gaining increasing interest as a marker of intrathecal immunoglobulin synthesis. However, data on inter-laboratory agreement of these measures is lacking. The aim was to assess the concordance of CSF and serum KFLC measurements, and of K-index values, across different laboratories. KFLC and albumin of 15 paired CSF and serum samples were analyzed by eight participating laboratories. Four centers used Binding Site instruments and assays (B), three used Siemens instruments and assays (S), and one center used a Siemens instrument with a Binding Site assay (mixed). Absolute individual agreement was calculated using a two-way mixed effects intraclass correlation coefficient (ICC). Cohen's kappa coefficient (k) was used to measure agreement on positive (≥5.8) K-index values. There was an excellent agreement in CSF KFLC measurements across all laboratories (ICC (95% confidence interval): 0.93 (0.87-0.97)) and of serum KFLC across B and S laboratories (ICC: 0.91 (0.73-0.97)), while ICC decreased (to 0.81 (0.53-0.93)) when including the mixed laboratory in the analysis. Concordance for a positive K-Index was substantial across all laboratories (k = 0.77) and within S laboratories (k = 0.71), and very good (k = 0.89) within B laboratories, meaning that patients rarely get discordant results on K-index positivity notwithstanding the testing in different laboratories and the use of different platforms/assays.

We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-β1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro.

Cornification is a specialized mode of the cell-death program exclusively allowed for terrestrial amniotes. Recent investigations suggest that loricrin (LOR) is an important cornification effector. As the connotation of its name ("lorica" meaning an armor in Latin) suggests, the keratin-associated protein LOR promotes the maturation of the epidermal structure through organizing covalent cross-linkages, endowing the epidermis with the protection against oxidative injuries. By reviewing cornification mechanisms, we seek to classify ichthyosiform dermatoses based on their function, rather than clinical manifestations. We also reviewed recent mechanistic insights into the Kelch-like erythroid cell-derived protein with the cap "n" collar homology-associated protein 1/nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway in skin health and diseases, as LOR and NRF2 coordinate the epidermis-intrinsic xenobiotic metabolism. Finally, we refine the theoretical framework of cross-talking between keratinocytes and epidermal resident leukocytes, dissecting an LOR immunomodulatory function.

BACKGROUND: Gastric cancer (GC) is one of the leading malignant diseases worldwide, especially in Asia. CAST is a potential oncogene in GC carcinogenesis. The character of macrophage infiltration in the GC microenvironment also remains unaddressed. METHODS: We first applied machine searching to evaluate gene candidates for GC. CAST expression and pan-cancer surveyance were analyzed using the Human Protein Atlas (HPA) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. The protein-protein interaction (PPI) network was downloaded from STRING. We investigated the impact of CAST on clinical prognosis using a Kaplan-Meier plotter. The correlations between CAST and Lgr5 and macrophage infiltration in GC were determined using TIMER 2.0. Finally, GeneMANIA was also used to evaluate the possible functional linkages between genes. RESULTS: After the machine-assisted search, CAST expression was found to significantly influence the overall survival of GC patients. STRING revealed CAST-related proteomic and transcriptomic associations, mainly concerning the CAPN family. Moreover, CAST significantly impacts the prognosis of GC based on the validation of other datasets. Notably, high CAST expression was correlated with worse overall survival in GC patients (hazard ratio = 1.59; log-rank P = 9.4 × 10-8). CAST and Lgr5 expression were both positively correlated with WNT 2 and WNT 2B. Among the GC patients in several datasets, CAST and macrophage infiltration, evaluated together, showed no obvious association with poor clinical overall survival. CONCLUSIONS: CAST plays an important role in the clinical prognosis of GC and is associated with WNT 2/WNT 2B/Lgr5. Our study demonstrates that CAST's influence on overall survival in GC is regulated by macrophage infiltration.

Th17 cells are implicated in allergic inflammatory diseases, including allergic rhinitis (AR), though the effect of steroids on Th17 cell-dependent nasal responses is unclear. Herein, we investigated a nasal inflammation model elicited by allergen provocation in mice infused with Th17 cells and its responsiveness against steroid treatment. We transferred BALB/c mice with Th17 cells, which were differentiated in vitro and showed a specific reaction to ovalbumin (OVA). We challenged the transferred mice by intranasal injection of OVA and to some of them, administered dexamethasone (Dex) subcutaneously in advance. Then, we assessed immediate nasal response (INR), nasal hyperresponsiveness (NHR), and inflammatory cell infiltration into the nasal mucosa. The significant nasal inflammatory responses with massive neutrophil accumulation, INR, and NHR were induced upon allergen challenge. Allergen-induced INR and NHR were significantly suppressed by Dex treatment. This study suggested the effectiveness of steroids on Th17 cell-mediated nasal responses in AR.

The transcription factor EB (TFEB) is a master regulator of lysosomal function and autophagy. Mechanistic target of rapamycin (mTOR)-mediated phosphorylation on TFEB is known to regulate TFEB subcellular localization and activity at the lysosomal surface. Recent studies have shown that TFEB also plays a critical role in physiological processes such as lipid metabolism, and dysfunction of TFEB has been observed in the pathogenesis of several diseases. Owing to its ability to improve disease status in murine models, TFEB has attracted attention as a therapeutic target for diseases. In this review, we will present the regulation of TFEB and its role in the pathogenesis of liver diseases, particularly non-alcoholic fatty liver disease (NAFLD).

Human adenylate kinase 1 (hAK1) plays a vital role in the energetic and metabolic regulation of cell life, and impaired functions of hAK1 are closely associated with many diseases. In the presence of Mg2+ ions, hAK1 in vivo can catalyze two ADP molecules into one ATP and one AMP molecule, activating the downstream AMP signaling. The ADP-binding also initiates AK1 transition from an open conformation to a closed conformation. However, how substrate binding triggers the conformational transition of hAK1 is still unclear, and the underlying molecular mechanisms remain elusive. Herein, we determined the solution structure of apo-hAK1 and its key residues for catalyzing ADP, and characterized backbone dynamics characteristics of apo-hAK1 and hAK1-Mg2+-ADP complex (holo-hAK1) using NMR relaxation experiments. We found that ADP was primarily bound to a cavity surrounded by the LID, NMP, and CORE domains of hAK1, and identified several critical residues for hAK1 catalyzing ADP including G16, G18, G20, G22, T39, G40, R44, V67, D93, G94, D140, and D141. Furthermore, we found that apo-hAK1 adopts an open conformation with significant ps-ns internal mobility, and Mg2+-ADP binding triggered conformational transition of hAK1 by suppressing the ps-ns internal motions of α3α4 in the NMP domain and α7α8 in the LID domain. Both α3α4 and α7α8 fragments became more rigid so as to fix the substrate, while the catalyzing center of hAK1 experiences promoted µs-ms conformational exchange, potentially facilitating catalysis reaction and conformational transition. Our results provide the structural basis of hAK1 catalyzing ADP into ATP and AMP, and disclose the driving force that triggers the conformational transition of hAK1, which will deepen understanding of the molecular mechanisms of hAK1 functions.

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP-ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at -196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells.

Liver cancer stem cells (LCSCs) are a small subset of oncogenic cells with a self-renewal ability and drug resistance, and they promote the recurrence and metastasis of hepatocellular carcinoma (HCC). However, the mechanisms regulating LCSCs have not been fully explored. By enriching LCSCs from spheroid cultures and performing transcriptomic analysis, we determined that alanine-glyoxylate aminotransferase (AGXT), which participates in the metabolism of serine and glycine, was significantly upregulated in spheroid cultures, and its function in LCSCs remains unknown. Through the exogenous overexpression or short hairpin RNA knockdown of AGXT in HCC cells, we observed that changes in the AGXT level did not affect the spheroid ability and population of LCSCs. The knockdown of AGXT in LCSCs reduced the number of spheroids and the population of LCSCs; this implies that AGXT is required for the maintenance of cancer stemness rather than as a driver of LCSCs. Mechanistically, AGXT may sustain the self-renewal potential of LCSCs by upregulating the expression of SRY-box transcription factor 2 (SOX2) and octamer-binding transcription factor 4 (OCT4), two well-known master regulators of cancer stemness. Taken together, our study demonstrates the role of AGXT in supporting LCSCs; thus, AGXT merits further exploration.

Palmitoylethanolamide (PEA) stands out among endogenous lipid mediators for its neuroprotective, anti-inflammatory, and analgesic functions. PEA belonging to the N-acetylanolamine class of phospholipids was first isolated from soy lecithin, egg yolk, and peanut flour. It is currently used for the treatment of different types of neuropathic pain, such as fibromyalgia, osteoarthritis, carpal tunnel syndrome, and many other conditions. The properties of PEA, especially of its micronized or ultra-micronized forms maximizing bioavailability and efficacy, have sparked a series of innovative research to evaluate its possible application as therapeutic agent for neurodegenerative diseases. Neurodegenerative diseases are widespread throughout the world, and although they are numerous and different, they share common patterns of conditions that result from progressive damage to the brain areas involved in mobility, muscle coordination and strength, mood, and cognition. The present review is aimed at illustrating in vitro and in vivo research, as well as human studies, using PEA treatment, alone or in combination with other compounds, in the presence of neurodegeneration. Namely, attention has been paid to the effects of PEA in counteracting neuroinflammatory conditions and in slowing down the progression of diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Frontotemporal dementia, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis. Literature research demonstrated the efficacy of PEA in addressing the damage typical of major neurodegenerative diseases.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Astrocytes are the most abundant cells in the CNS parenchyma and play an essential role in several brain functions, such as the fine-tuning of synaptic transmission, glutamate uptake and the modulation of immune responses, among others. Much of the knowledge on the biology of astrocytes has come from the study of rodent primary astrocytic cultures. Usually, the culture is a mixed population of astrocytes and a small proportion of microglia. However, it is critical to have a pure culture of astrocytes if one wants to address their inflammatory response. If present, microglia sense the stimulus, rapidly proliferate and react to it, making it unfeasible to assess the individual responsiveness of astrocytes. Microglia have been efficiently eliminated in vivo through PLX-3397, a colony-stimulating factor-1 receptor (CSF-1R) inhibitor. In this work, the effectiveness of PLX-3397 in eradicating microglia from primary mixed glial cultures was evaluated. We tested three concentrations of PLX-3397-0.2 μM, 1 μM and 5 μM-and addressed its impact on the culture yield and viability of astrocytes. PLX-3397 is highly efficient in eliminating microglia without affecting the viability or response of cultured astrocytes. Thus, these highly enriched monolayers of astrocytes allow for the more accurate study of their immune response in disease conditions.

In adults, glucocorticoids are stress hormones that act, partly, through actions on mitochondrial oxidative phosphorylation (OXPHOS) to increase energy availability. Before birth, glucocorticoids are primarily maturational signals that prepare the fetus for new postnatal challenges. However, the role of the normal prepartum glucocorticoid rise in preparing mitochondria for the increased postnatal energy demands remains largely unknown. This study examined the effect of physiological increases in the fetal cortisol concentration on cerebral mitochondrial OXPHOS capacity near term (~130 days gestation, term ~145 days gestation). Fetal sheep were infused with saline or cortisol for 5 days at ~0.8 of gestation before the mitochondrial content, respiratory rates, abundance of the electron transfer system proteins and OXPHOS efficiency were measured in their cortex and cerebellum. Cerebral morphology was assessed by immunohistochemistry and stereology. Cortisol treatment increased the mitochondrial content, while decreasing Complex I-linked respiration in the cerebellum. There was no effect on the cortical mitochondrial OXPHOS capacity. Cortisol infusion had regional effects on cerebral morphology, with increased myelination in the cerebrum. The findings demonstrate the importance of cortisol in regulating the cerebral mitochondrial OXPHOS capacity prenatally and have implications for infants born preterm or after glucocorticoid overexposure due to pregnancy complications or clinical treatment.

As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers' design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a]2-SHa and [G10a]3-SHa, and [G10a]2-SHa conjugated with a polymer molecule, i.e., Jeff-[G10a]2-SHa, were synthesized, purified on RP-HPLC and UPLC and fully characterized by mass, NMR spectroscopic techniques, circular dichroism, ultraviolet, infrared, dynamic light scattering, and atomic force microscopic studies. In pH- and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40-60 °C and pH values in the range of 6-12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs (breast, lung, prostate, pancreas, and liver). The high hemolytic activity of [G10a]2-SHa and [G10a]3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a]2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.

Pancreatic cancer represents one of the leading causes of oncological death worldwide. A combination of pancreatic cancer aggressiveness and late diagnosis are key factors leading to a low survival rate and treatment inefficiency, and early diagnosis is pursued as a critical factor for pancreatic cancer. In this context, plasma microRNAs are emerging as promising players due to their non-invasive and practical usage in oncological diagnosis and prognosis. Recent studies have showed some miRNAs associated with pancreatic cancer subtypes, or with stages of the disease. Here we demonstrate plasma exosome-derived microRNA expression in pancreatic cancer patients and healthy individuals from Brazilian patients. Using plasma of 65 pancreatic cancer patients and 78 healthy controls, plasma exosomes were isolated and miRNAs miR-27b, miR-125b-3p, miR-122-5p, miR-21-5p, miR-221-3p, miR-19b, and miR-205-5p were quantified by RT-qPCR. We found that miR-125b-3p, miR-122-5p, and miR-205-5p were statistically overexpressed in the plasma exosomes of pancreatic cancer patients compared to healthy controls. Moreover, miR-205-5p was significantly overexpressed in European descendants, in patients with tumor progression and in those who died from the disease, and diagnostic ability by ROC curve was 0.86. Therefore, we demonstrate that these three microRNAs are potential plasma exosome-derived non-invasive biomarkers for the diagnosis and prognosis of Brazilian pancreatic cancer, demonstrating the importance of different populations and epidemiological bias.

Sex steroids, derived mainly from gonads, can shape microbiota composition; however, the impact of gonadectomy and sex on steroid production in the gut (i.e., gut steroids), and its interaction with microbiota composition, needs to be clarified. In this study, steroid environment and gut steroidogenesis were analysed by liquid chromatography tandem mass spectrometry and expression analyses. Gut microbiota composition as branched- and short-chain fatty acids were determined by 16S rRNA gene sequence analysis and gas chromatography flame ionisation detection, respectively. Here, we first demonstrated that levels of pregnenolone (PREG), progesterone (PROG), and isoallopregnanolone (ISOALLO) were higher in the female rat colon, whereas the level of testosterone (T) was higher in males. Sexual dimorphism on gut steroidogenesis is also reported after gonadectomy. Sex, and more significantly, gonadectomy, affects microbiota composition. We noted that a number of taxa and inferred metabolic pathways were associated with gut steroids, such as positive associations between Blautia with T, dihydroprogesterone (DHP), and allopregnanolone (ALLO), whereas negative associations were noted between Roseburia and T, ALLO, PREG, ISOALLO, DHP, and PROG. In conclusion, this study highlights the novel sex-specific association between microbiota and gut steroids with possible relevance for the gut-brain axis.

Nuclear-factor-E2-related factor 2 (Nrf2) is a key transcription factor for the regulation of cellular responses to cellular stress and inflammation, and its expression is significantly lower after spontaneous term labor in human fetal membranes. Pathological induction of inflammation can lead to adverse pregnancy outcomes such as pre-eclampsia, preterm labor, and fetal death. As stretch forces are known to act upon the fetal membranes in utero, we aimed to ascertain the effect of stretch on Nrf2 to increase our understanding of the role of this stimulus on cells of the amnion at term. Our results indicated a significant reduction in Nrf2 expression in stretched isolated human amnion epithelial cells (hAECs) that could be rescued with sulforaphane treatment. Downregulation of Nrf2 as a result of stretch was accompanied with activation of proinflammatory nuclear factor-kB (NF-kB) and increases in LDH activity, ROS, and HMGB1. This work supports stretch as a key modulator of cellular stress and inflammation in the fetal membranes. Our results showed that the modulation of the antioxidant response pathway in the fetal membranes through Nrf2 activation may be a viable approach to improve outcomes in pregnancy.

In the present study, we evaluated the association of the BAT1, NFKBIL, LTA, and CASP1 single nucleotide polymorphisms and the gene-gene interactions with risk of developing restenosis after coronary stenting. The allele and genotype determination of the polymorphisms (BAT1 rs2239527 C/G, NFKBIL1 rs2071592 T/A, LTA rs1800683 G/A, CASP1 rs501192 A/G, and CASP1 rs580253 A/G) were performed by 5'exonuclease TaqMan assays in 219 patients: 66 patients with restenosis and 153 without restenosis. The distribution of rs2239527 C/G, rs2071592 T/A, and rs1800683 G/A polymorphisms was similar in patients with and without restenosis. Nonetheless, under recessive (OR = 2.73, pCRes = 0.031) and additive models (OR = 1.65, pCAdd = 0.039), the AA genotype of the rs501192 A/G polymorphism increased the restenosis risk. Under co-dominant, dominant, recessive, and additive models, the AA genotype of the rs580253 A/G was associated with a high restenosis risk (OR = 5.38, pCCo-Dom = 0.003; OR = 2.12, pCDom = 0.031; OR = 4.32, pCRes = 0.001; and OR = 2.16, 95%CI: 1.33-3.52, pCAdd = 0.001, respectively). In addition, we identified an interaction associated with restenosis susceptibility: BAT1-NFKBIL1-LTA-CASP1 (OR = 9.92, p < 0.001). In summary, our findings demonstrate that the rs501192 A/G and rs580253 A/G polymorphisms, as well as the gene-gene interactions between BAT1-NFKBIL1-LTA-CASP1, are associated with an increased restenosis risk after coronary stenting.

The discovery of aldehydes dates back to 1774 when Carl Wilhelm Scheele synthesized acetaldehyde [...].

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Sigma-1 receptor (S1R) is an important endoplasmic reticulum chaperone with various functions in health and disease. The purpose of the current work was to elucidate the involvement of S1R in cancer energy metabolism under its basal, activated, and inactivated states. For this, two cancer cell lines that differentially express S1R were treated with S1R agonist, (+)-SKF10047, and antagonist, BD1047. The effects of the agonist and antagonist on cancer energy metabolism were studied using single-cell fluorescence microscopy analysis of real-time ion and metabolite fluxes. Our experiments revealed that S1R activation by agonist increases mitochondrial bioenergetics of cancer cells while decreasing their reliance on aerobic glycolysis. S1R antagonist did not have a major impact on mitochondrial bioenergetics of tested cell lines but increased aerobic glycolysis of S1R expressing cancer cell line. Our findings suggest that S1R plays an important role in cancer energy metabolism and that S1R ligands can serve as tools to modulate it.

BACKGROUND: Aging society faces significant health challenges, among which cognitive-related disorders are emerging. Diet quality has been recognized among the major contributors to the rising prevalence of cognitive disorders, with increasing evidence of the putative role of plant-based foods and their bioactive components, including polyphenols. Dietary polyphenols, including phytoestrogens, have been hypothesized to exert beneficial effects toward brain health through various molecular mechanisms. However, the evidence on the association between dietary phytoestrogen intake and cognitive function is limited. The aim of this study was to investigate the association between phytoestrogen intake and cognitive status in a cohort of older adults living in Sicily, Southern Italy. METHODS: Dietary information from 883 individuals aged 50 years or older was collected through a validated food frequency questionnaire. Cognitive status was assessed through the Short Portable Mental Status Questionnaire. RESULTS: The highest total isoflavone (including daidzein and genistein) intake was inversely associated with cognitive impairment compared to the lowest (odds ratio (OR) = 0.43, 95% confidence interval (CI): 0.20-0.92). Higher intake of total lignans and, consistently, all individual compounds (with the exception of secoisolariciresinol) were inversely associated with cognitive impairment only in the unadjusted model. CONCLUSIONS: A higher intake of phytoestrogens, especially isoflavones, was associated with a better cognitive status in a cohort of older Italian individuals living in Sicily. Taking into account the very low intake of isoflavones in Italian diets, it is noteworthy to further investigate selected populations with habitual consumption of such compounds to test whether these results may be generalized to the Italian population.

Allostery-regulation at distant sites is a key concept in biology. The proteasome exhibits multiple forms of allosteric regulation. This regulatory communication can span a distance exceeding 100 Ångstroms and can modulate interactions between the two major proteasome modules: its core particle and regulatory complexes. Allostery can further influence the assembly of the core particle with regulatory particles. In this focused review, known and postulated interactions between these proteasome modules are described. Allostery may explain how cells build and maintain diverse populations of proteasome assemblies and can provide opportunities for therapeutic interventions.

Recently, the endocannabinoid system has attracted growing attention from the scientific community for its involvement in homeostatic and pathological processes as they pertains to human physiology. Among the constituents of the endocannabinoid system, the molecule palmitoyl ethanolamide has particularly been studied for its ability to reduce several inflammatory processes involving the central nervous system. Here, we reviewed published literature and summarized the main targets of the palmitoyl ethanolamide, along with its unique possible mechanisms for restoring correct functioning of the central nervous system. Moreover, we have highlighted a less-known characteristic of palmitoyl ethanolamide, namely its ability to modulate the function of the neuromuscular junction by binding to acetylcholine receptors in different experimental conditions. Indeed, there are several studies that have highlighted how ultra-micronized palmitoyl ethanolamide is an interesting nutraceutical support for the treatment of pathological neuromuscular conditions, specifically when the normal activity of the acetylcholine receptor is altered. Although further multicentric clinical trials are needed to confirm the efficacy of ultra-micronized palmitoyl ethanolamide in improving symptoms of neuromuscular diseases, all the literature reviewed here strongly supports the ability of this endocannabinoid-like molecule to modulate the acetylcholine receptors thus resulting as a valid support for the treatment of human neuromuscular diseases.

The peritoneal membrane is the largest internal membrane of the human body, having a surface area that approximates the surface area of the skin [...].

Bacterial resistance against antibiotics has led to increasing numbers of treatment failures, and AMPs are widely accepted as becoming potential alternatives due to their advantages. Temporin-PKE is a novel peptide extracted from the skin secretion of Pelophylax kl. esculentus and it displays a strong activity against Gram-positive bacteria, with an extreme cytotoxicity. Incorporating positively charged residues and introducing D-amino acids were the two main strategies adopted for the modifications. The transformation of the chirality of Ile could reduce haemolytic activity, and an analogue with appropriate D-isoforms could maintain antimicrobial activity and stability. The substitution of hydrophobic residues could bring about more potent and broad-spectrum antimicrobial activities. The analogues with Lys were less harmful to the normal cells and their stabilities remained at similarly high levels compared to temporin-PKE. The optimal number of charges was three, and the replacement on the polar face was a better choice. Temporin-PKE-3K exerted dually efficient functions includingstrong antimicrobial and anticancer activity. This analogue showed a reduced possibility for inducing resistance in MRSA and Klebsiella pneumoniae, a rather strong antimicrobial activity in vivo, and it exhibited the highest therapeutic index such that temporin-PKE-3K has the potential to be developed as a clinical drug.

A mycelial culture of the African basidiomycete Echinochaete cf. brachypora was studied for biologically active secondary metabolites, and four compounds were isolated from its crude extract derived from shake flask fermentations, using preparative high-performance liquid chromatography (HPLC). The pure metabolites were identified using extensive nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Aside from the new metabolites 1-methoxyneomarinone (1) and (E)-3-methyl-5-(-12,13,14-trimethylcyclohex-10-en-6-yl)pent-2-enoic acid (4), the known metabolites neomarinone (2) and fumaquinone (4) were obtained. Such compounds had previously only been reported from Actinobacteria but were never isolated from the cultures of a fungus. This observation prompted us to evaluate whether the above metabolites may actually have been produced by an endosymbiontic bacterium that is associated with the basidiomycete. We have indeed been able to characterize bacterial 16S rDNA in the fungal mycelia, and the production of the metabolites stopped when the fungus was sub-cultured on a medium containing antibacterial antibiotics. Therefore, we have found strong evidence that compounds 1-4 are not of fungal origin. However, the endofungal bacterium was shown to belong to the genus Ralstonia, which has never been reported to produce similar metabolites to 1-4. Moreover, we failed to obtain the bacterial strain in pure culture to provide final proof for its identity. In any case, the current report is the first to document that polyporoid Basidiomycota are associated with endosymbionts and constitutes the first report on secondary metabolites from the genus Echinochaete.

The anti-diabetic drug metformin is currently tested for the treatment of hematological and solid cancers. Proteasome inhibitors, e.g., Bortezomib, are approved for the treatment of multiple myeloma and mantle cell lymphoma but are also studied for lung cancer therapy. We here analyzed the interaction of the two drugs in two cell lines, namely the mantle cell lymphoma Jeko-1 and the non-small-cell lung cancer (NSCLC) H1299 cells, using proliferation and survival assays, native-gel analysis for proteasome activity and assembly, and expression analysis of proteasome assembly factors. Our results demonstrate that metformin treatment induces resistance of cancer cells to the proteasome inhibitor Bortezomib by impairing the activity and assembly of the 26S proteasome complexes. These effects of metformin on proteasome inhibitor sensitivity in cancer cells are of potential relevance for patients that receive proteasome inhibitor therapy.

Unconventional T cells and innate lymphoid cells (ILCs) make up a heterogeneous set of cells that characteristically show prompt responses toward specific antigens. Unconventional T cells recognize non-peptide antigens, which are bound and presented by diverse non-polymorphic antigen-presenting molecules and comprise γδ T cells, MR1-restricted mucosal-associated invariant T cells (MAITs), and natural killer T cells (NKTs). On the other hand, ILCs lack antigen-specific receptors and act as the innate counterpart to the T lymphocytes found in the adaptive immune response. The alteration of unconventional T cells and ILCs in frequency and functionality is correlated with the onset of several autoimmune diseases, allergy, inflammation, and tumor. However, depending on the physio-pathological framework, unconventional T cells may exhibit either protective or pathogenic activity in a range of neoplastic diseases. Nonetheless, experimental models and clinical studies have displayed that some unconventional T cells are potential therapeutic targets, as well as prognostic and diagnostic markers. In fact, cell-mediated immune response in tumors has become the focus in immunotherapy against neoplastic disease. This review concentrates on the present knowledge concerning the function of unconventional T cell sets in the antitumor immune response in hematological malignancies, such as acute and chronic leukemia, multiple myeloma, and lymphoproliferative disorders. Moreover, we discuss the possibility that modulating the activity of unconventional T cells could be useful in the treatment of hematological neoplasms, in the prevention of specific conditions (such as graft versus host disease), and in the formulation of an effective anticancer vaccine therapy. The exact knowledge of the role of these cells could represent the prerequisite for the creation of a new form of immunotherapy for hematological neoplasms.

Ferroverdins are ferrous iron (Fe2+)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe2+, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe2+-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

The ubiquitin-proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.

Targeted therapy uses multiple ways of ensuring that the drug will be delivered to the desired site. One of these ways is an encapsulation of the drug and functionalization of the surface. Among the many molecules that can perform such a task, the present work focused on the antibodies of single-chain variable fragments (scFvs format). We studied scFv, which specifically recognizes the dopamine D2 and serotonin 5-HT1A receptor heteromers. The scFvD2-5-HT1A protein was analyzed biochemically and biologically, and the obtained results indicated that the antibody is properly folded and non-toxic and can be described as low-immunogenic. It is not only able to bind to the D2-5-HT1A receptor heteromer, but it also influences the cAMP signaling pathway and-when surfaced on nanogold particles-it can cross the blood-brain barrier in in vitro models. When administered to mice, it decreased locomotor activity, matching the effect induced by clozapine. Thus, we are strongly convinced that scFvD2-5-HT1A, which was a subject of the present investigation, is a promising targeting ligand with the potential for the functionalization of nanocarriers targeting selected areas of the brain.

CircRNAs play an important role in fat deposition, and testosterone-deficient boars exhibit significantly increased fat deposition; however, the mechanism by which testosterone regulates fat deposition through circRNAs remains unclear. In this study, circRNA-seq of backfat and abdominal fat from castrated and intact full-sib Yorkshire pigs was performed. The GO and KEGG enrichment analyses revealed that the host genes of the dorsal DE circRNAs were mainly involved in fatty acid transport, while in abdominal tissues, these genes were mainly involved in adipogenesis and inflammation. The interaction among sus_circPAPPA2, ssc-miR-2366 and GK was verified by dual fluorescence experiments and in porcine preadipocytes. The overexpression of sus_circPAPPA2 significantly inhibited the differentiation of preadipocytes. The expression of sus_circPAPPA2 was increased after adding 100 nM of testosterone, and preadipocyte differentiation was significantly inhibited. Testosterone can affect preadipocyte differentiation by upregulating the expression of sus_circPAPPA2, sponging miR-2366 and regulating the expression of genes, such as GK. These results indicate that testosterone can regulate the expression of adipocyte differentiation- and lipid metabolism-related genes by regulating the expression of circRNA, and ceRNA networks are different in the testosterone regulation of adipose deposition in different parts. This study provides basic data enhancing the understanding of the interaction between the hormone environment and mir-2366/GK to regulate trait performance in pigs.

There is a growing prevalence of vascular cognitive impairment (VCI) worldwide, and most research has suggested that cerebral small vessel disease (CSVD) is the main contributor to VCI. Several potential physiopathologic mechanisms have been proven to be involved in the process of CSVD, such as blood-brain barrier damage, small vessels stiffening, venous collagenosis, cerebral blood flow reduction, white matter rarefaction, chronic ischaemia, neuroinflammation, myelin damage, and subsequent neurodegeneration. However, there still is a limited overall understanding of the sequence and the relative importance of these mechanisms. The glymphatic system (GS) and meningeal lymphatic vessels (mLVs) are the analogs of the lymphatic system in the central nervous system (CNS). As such, these systems play critical roles in regulating cerebrospinal fluid (CSF) and interstitial fluid (ISF) transport, waste clearance, and, potentially, neuroinflammation. Accumulating evidence has suggested that the glymphatic and meningeal lymphatic vessels played vital roles in animal models of CSVD and patients with CSVD. Given the complexity of CSVD, it was significant to understand the underlying interaction between glymphatic and meningeal lymphatic transport with CSVD. Here, we provide a novel framework based on new advances in main four aspects, including vascular risk factors, potential mechanisms, clinical subtypes, and cognition, which aims to explain how the glymphatic system and meningeal lymphatic vessels contribute to the progression of CSVD and proposes a comprehensive insight into the novel therapeutic strategy of CSVD.

Nitric oxide (NO) is a potent signaling molecule involved in many physiological and pathophysiological processes in the kidney. NO plays a complex role in glomerular ultrafiltration, vasodilation, and inflammation. Changes in NO bioavailability in pathophysiological conditions such as hypertension or diabetes may lead to podocyte damage, proteinuria, and rapid development of chronic kidney disease (CKD). Despite the extensive data highlighting essential functions of NO in health and pathology, related signaling in glomerular cells, particularly podocytes, is understudied. Several reports indicate that NO bioavailability in glomerular cells is decreased during the development of renal pathology, while restoring NO level can be beneficial for glomerular function. At the same time, the compromised activity of nitric oxide synthase (NOS) may provoke the formation of peroxynitrite and has been linked to autoimmune diseases such as systemic lupus erythematosus. It is known that the changes in the distribution of NO sources due to shifts in NOS subunits expression or modifications of NADPH oxidases activity may be linked to or promote the development of pathology. However, there is a lack of information about the detailed mechanisms describing the production and release of NO in the glomerular cells. The interaction of NO and other reactive oxygen species in podocytes and how NO-calcium crosstalk regulates glomerular cells' function is still largely unknown. Here, we discuss recent reports describing signaling, synthesis, and known pathophysiological mechanisms mediated by the changes in NO homeostasis in the podocyte. The understanding and further investigation of these essential mechanisms in glomerular cells will facilitate the design of novel strategies to prevent or manage health conditions that cause glomerular and kidney damage.

Idiopathic Parkinson's disease (iPD) is characterized by degeneration of the dopaminergic substantia nigra pars compacta (SNc), typically in the presence of Lewy pathology (LP) and mitochondrial respiratory complex I (CI) deficiency. LP is driven by α-synuclein aggregation, morphologically evolving from early punctate inclusions to Lewy bodies (LBs). The relationship between α-synuclein aggregation and CI deficiency in iPD is poorly understood. While studies in models suggest they are causally linked, observations in human SNc show that LBs preferentially occur in CI intact neurons. Since LBs are end-results of α-synuclein aggregation, we hypothesized that the relationship between LP and CI deficiency may be better reflected in neurons with early-stage α-synuclein pathology. Using quadruple immunofluorescence in SNc tissue from eight iPD subjects, we assessed the relationship between neuronal CI or CIV deficiency and early or late forms of LP. In agreement with previous findings, we did not observe CI-negative neurons with late LP. In contrast, early LP showed a significant predilection for CI-negative neurons (p = 6.3 × 10-5). CIV deficiency was not associated with LP. Our findings indicate that early α-syn aggregation is associated with CI deficiency in iPD, and suggest a double-hit mechanism, where neurons exhibiting both these pathologies are selectively lost.

Galectins are a family of soluble β-galactoside-binding proteins with diverse glycan-dependent and glycan-independent functions outside and inside the cell [...].

Update of bioRxiv. 2020 Mar 08;:

Gadolinium (Gd) is one of the rare-earth elements. The properties of its trivalent cation (Gd3+) make it suitable to serve as the central ion in chelates administered intravenously to patients as a contrast agent in magnetic resonance imaging. Such Gd-chelates have been used for more than thirty years. During the past decades, knowledge has increased about potential harmful effects of Gd-chelates in patients with severe renal dysfunction. In such patients, there is a risk for a potentially disabling and lethal disease, nephrogenic systemic fibrosis. Restricting the use of Gd-chelates in persons with severely impaired renal function has decreased the occurrence of this toxic effect in the last decade. There has also been an increasing awareness of Gd-retention in the body, even in patients without renal dysfunction. The cumulative number of doses given, and the chemical structure of the chelate given, are factors of importance for retention in tissues. This review describes the chemical properties of Gd and its medically used chelates, as well as its toxicity and potential side effects related to injection of Gd-chelates.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic insulin resistance (IR) in obesity are still unclear. Here, we sought to determine the effects of a high fat (HF) diet supplemented with MA on obesity-associated metabolic disorders in mice. Wild-type C57BL/6 mice were fed a HF diet in the presence or absence of 3% MA for 12 weeks. Plasma lipids, plasma adipokines, AT inflammation, systemic IR, glucose homeostasis, and hepatic steatosis were assessed. The body weight and visceral adipose tissue (VAT) mass were significantly higher in mice receiving the HF+MA diet compared to HF diet-fed controls. Plasma total cholesterol levels were marginally increased in HF+MA-fed mice compared to controls. Fasting blood glucose was comparable between HF and HF+MA-fed mice. Interestingly, the plasma insulin and HOMA-IR index, a measure of insulin resistance, were significantly higher in HF+MA-fed mice compared to HF controls. Macrophage and inflammatory markers were significantly elevated in the AT and AT-derived stromal vascular cells upon MA feeding. Moreover, the level of circulating resistin, an adipokine promoting insulin resistance, was significantly higher in HF+MA-fed mice compared with HF controls. The insulin tolerance test revealed that the IR was higher in mice receiving the MA supplementation compared to HF controls. Moreover, the glucose tolerance test showed impairment in systemic glucose homeostasis in MA-fed mice. Analyses of liver samples showed a trend towards an increase in liver TG upon MA feeding. However, markers of oxidative stress and inflammation were reduced in the liver of mice fed an MA diet compared to controls. Taken together, our data suggest that chronic administration of MA in diet exacerbates obesity-associated insulin resistance and this effect is mediated in part, via increased AT inflammation and increased secretion of resistin.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disorder that particularly affects the salivary and lachrymal glands, generally causing a typical dryness of the eyes and of the mouth. The disease encompasses diverse clinical representations and is characterized by B-cell polyclonal activation and autoantibodies production, including anti-Ro/SSA. Recently, it has been suggested that autoantibody profiling may enable researchers to identify susceptible asymptomatic individuals in a pre-disease state. In this pilot study, we used mass spectrometry to analyze and compare the salivary proteomics of patients with established pSS and patients with pre-clinical SS, identifying a common protein signature in their salivary fluid. We found that several inflammatory, immunity-related, and typical acinar proteins (such as MUC5B, PIP, CST4, and lipocalin 1) were differently expressed in pSS and in pre-clinical SSA+ carriers, compared to healthy controls. This suggests that saliva may closely reflect exocrine gland inflammation from the early phases of the disease. This study confirms the value of salivary proteomics for the identification of reliable biomarkers for SS that could be identified, even in a preclinical phase of the disease.

A complex physiological culture medium (Plasmax) was introduced recently, composed of nutrients and metabolites at concentrations normally found in human plasma to mimic the in vivo environment for cell line cultivation. As glycosylation has been proved to be involved in cancer development, it is necessary to investigate the glycan expression changes in media with different nutrients. In this study, a breast cancer cell line, MDA-MB-231BR, and a brain cancer cell line, CRL-1620, were cultivated in Plasmax and commercial media to reveal cell line glycosylation discrepancies prompted by nutritional environments. Glycomics analyses of cell lines were performed using LC-MS/MS. The expressions of multiple fucosylated N-glycans, such as HexNAc4Hex3DeoxyHex1 and HexNAc5Hex3DeoxyHex1, derived from both cell lines exhibited a significant increase in Plasmax. Among the O-glycans, significant differences were also observed. Both cell lines cultivated in EMEM had the lowest amounts of O-glycans expressed. The original work described the development of Plasmax, which improves colony formation, and resulted in transcriptomic and metabolomic alterations of cancer cell lines, while our results indicate that Plasmax can significantly impact protein glycosylation. This study also provides information to guide the selection of media for in vitro cancer cell glycomics studies.

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters.

Since first being documented in ancient times, the relation of inflammation with injury and disease has evolved in complexity and causality. Early observations supported a cause (injury) and effect (inflammation) relationship, but the number of pathologies linked to chronic inflammation suggests that inflammation itself acts as a potent promoter of injury and disease. Additionally, results from studies over the last 25 years point to chronic inflammation and innate immune signaling as a critical link between stress (exogenous and endogenous) and adaptation. This brief review looks to highlight the role of the innate immune response in disease pathology, and recent findings indicating the innate immune response to chronic stresses as an influence in driving adaptation.

Post-traumatic stress disorder (PTSD) is a trauma-related disorder. Platelet monoamine oxidase (MAO-B) is a peripheral biomarker associated with various symptoms in different psychopathologies, but its role in PTSD or different symptoms in PTSD is not clear. This study elucidated the association between platelet MAO-B activity and clinical symptoms occurring in PTSD. Platelet MAO-B activity was determined in 1053 male Caucasian subjects: 559 war veterans with PTSD (DSM-5 criteria), 62 combat exposed veterans who did not develop PTSD, and 432 non-combat exposed healthy controls. Clinical symptoms in PTSD were determined using CAPS and PANSS. Platelet MAO-B activity, controlled for the effect of smoking, was significantly increased in PTSD with severe versus mild and moderate traumatic symptoms, and was significantly decreased in PTSD subjects with severe versus mild positive, psychotic, and depressive symptoms. This finding was further confirmed with reduced platelet MAO-B activity in PTSD veterans with severe versus mild individual items of the PANSS-depressed, PANSS-psychotic, and PANSS-positive subscales. Altered platelet MAO-B activity, controlled for the possible confounders, was associated with the development and severity of different symptoms occurring in PTSD. These findings confirmed the role of platelet MAO-B activity as a peripheral marker of various psychopathological symptoms.

Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

The ventral tegmental area (VTA) in the ventral midbrain is the origin of the dopaminergic neurotransmission pathways. Although GABAA receptors and AKT-GSK3β signaling are involved in the pathophysiology of mental disorders and are modulated by antipsychotics, an unmet task is to reveal the pathological changes in these biomarkers and antipsychotic modulations in the VTA. Using a juvenile polyriboinosinic-polyribocytidylic acid (Poly I:C) psychiatric rat model, this study investigated the effects of adolescent risperidone treatment on GABAA receptors and AKT/GSK3β in the VTA. Pregnant female Sprague-Dawley rats were administered Poly I:C (5mg/kg; i.p) or saline at gestational day 15. Juvenile female offspring received risperidone (0.9 mg/kg, twice per day) or a vehicle from postnatal day 35 for 25 days. Poly I:C offspring had significantly decreased mRNA expression of GABAA receptor β3 subunits and glutamic acid decarboxylase (GAD2) in the VTA, while risperidone partially reversed the decreased GAD2 expression. Prenatal Poly I:C exposure led to increased expression of AKT2 and GSK3β. Risperidone decreased GABAA receptor β2/3, but increased AKT2 mRNA expression in the VTA of healthy rats. This study suggests that Poly I:C-elicited maternal immune activation and risperidone differentially modulate GABAergic neurotransmission and AKT-GSK3β signaling in the VTA of adolescent rats.

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A165 depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes-determined as a measure of permeability-was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A165 protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of β-catenin were only elevated on day two. Extracellular levels of VEGF-A-measured by ELISA-were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A165 to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled.

Homocysteine is a sulfur-containing endogenous amino acid leading to neurotoxic effects at high concentrations. Population studies suggest an association between plasma homocysteine levels and the risk of migraine headaches. The aim of this study was to analyze the sensitivity of rats with prenatal hyperhomocysteinemia (hHCY) in respect of the development of behavioral correlates of headache and spreading cortical depolarization (CSD) in a migraine model induced by the administration of the nitric oxide (NO) donor nitroglycerin. Animals with hHCY were characterized by migraine-related symptoms such as mechanical hyperalgesia, high-level anxiety, photophobia, as well as an enhanced level of neuronal activity in the somatosensory cortex along with a lower threshold of CSD generation. Likewise, acute or chronic intermittent administration of nitroglycerin also induced the development of mechanical allodynia, photophobia and anxiety in control groups. However, these symptoms were more pronounced in rats with hHCY. Unlike hHCY, nitroglycerin administration did not affect the threshold of CSD generation, but like hHCY, increased the background neuronal activity in layers 2/3 and 4 of the cerebral cortex. The latter was more pronounced in animals with hHCY. Thus, the migraine profile associated with hHCY can be further exaggerated in conditions with enhanced levels of migraine triggering the gaseous transmitter NO. Our data are consistent with the view that high levels of plasma homocysteine can act as a risk factor for the development of migraine.

Current management of heart failure (HF) is centred on modulating the progression of symptoms and severity of left ventricular dysfunction. However, specific understandings of genetic and molecular targets are needed for more precise treatments. To attain a clearer picture of this, we studied transcriptome changes in a chronic progressive HF model. Fifteen sheep (Ovis aries) underwent supracoronary aortic banding using an inflatable cuff. Controlled and progressive induction of pressure overload in the LV was monitored by echocardiography. Endomyocardial biopsies were collected throughout the development of LV failure (LVF) and during the stage of recovery. RNA-seq data were analysed using the PANTHER database, Metascape, and DisGeNET to annotate the gene expression for functional ontologies. Echocardiography revealed distinct clinical differences between the progressive stages of hypertrophy, dilatation, and failure. A unique set of transcript expressions in each stage was identified, despite an overlap of gene expression. The removal of pressure overload allowed the LV to recover functionally. Compared to the control stage, there were a total of 256 genes significantly changed in their expression in failure, 210 genes in hypertrophy, and 73 genes in dilatation. Gene expression in the recovery stage was comparable with the control stage with a well-noted improvement in LV function. RNA-seq revealed the expression of genes in each stage that are not reported in cardiovascular pathology. We identified genes that may be potentially involved in the aetiology of progressive stages of HF, and that may provide future targets for its management.

(1) Background: Abnormal repair after alveolar epithelial injury drives the progression of idiopathic pulmonary fibrosis (IPF). The maintenance of epithelial integrity is based on the self-renewal and differentiation of alveolar type 2 (AT2) cells, which require sufficient energy. However, the role of glutamine metabolism in the maintenance of the alveolar epithelium remains unclear. In this study, we investigated the role of glutamine metabolism in AT2 cells of patients with IPF and in mice with bleomycin-induced fibrosis. (2) Methods: Single-cell RNA sequencing (scRNA-seq), transcriptome, and metabolomics analyses were conducted to investigate the changes in the glutamine metabolic pathway during pulmonary fibrosis. Metabolic inhibitors were used to stimulate AT2 cells to block glutamine metabolism. Regeneration of AT2 cells was detected using bleomycin-induced mouse lung fibrosis and organoid models. (3) Results: Single-cell analysis showed that the expression levels of catalytic enzymes responsible for glutamine catabolism were downregulated (p < 0.001) in AT2 cells of patients with IPF, suggesting the accumulation of unusable glutamine. Combined analysis of the transcriptome (p < 0.05) and metabolome (p < 0.001) revealed similar changes in glutamine metabolism in bleomycin-induced pulmonary fibrosis in mice. Mechanistically, inhibition of the key enzymes involved in glucose metabolism, glutaminase-1 (GLS1) and glutamic-pyruvate transaminase-2 (GPT2) leads to reduced proliferation (p < 0.01) and differentiation (p < 0.01) of AT2 cells. (4) Conclusions: Glutamine metabolism is required for alveolar epithelial regeneration during lung injury.

Goat milk (GM) is an excellent alternative to cow milk and has recently been used in commercial infant formula preparation due to its superior fat composition. Here, the fatty acid (FA) composition, triacylglycerol (TAG) molecular species, thermal behavior and infrared spectra of extracted milk fat from the milk of the two main breeds of dairy goat bred in China (Guanzhong GM (GZG) and Xinong Saanen GM (XSG)) are investigated. Gas chromatography, Fourier-transform infrared spectroscopy, differential scanning calorimetry and ultra-performance convergence chromatography with quadrupole time-of-flight mass spectrometry are applied. The obtained results evidence significant fat compositional differences based on the breed that produced the considered GM. The major FAs in both GM fats were capric (C10:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1 n-9c). GZG presented a higher content of medium-chain saturated FAs, while XSG had higher unsaturated FAs with higher ratios of L/Ln and n-6/n-3. A total of 339 and 359 TAGs were detected and quantified in GZG and XSG, and the major TAGs were those of m/z 740.6712 (14.10 ± 0.27%) and m/z 684.6094 (10.94 ± 0.02%), respectively. Milk TAGs of GZG and XSG showed 24-54 and 26-54 total acyl carbon numbers with a 0-4 and 0-5 double bond number at 68 and 72 various retention times, respectively. Thermal analysis showed that all GM fat samples melted below normal body temperature. Infrared spectra revealed higher absorption values of GZG milk fat. This study provides valuable information to the dairy industry sector about GM fat produced in China, assessing the appropriateness of Chinese GM fat to be applied in Chinese infant formula.

Periodontitis is a destructive disease of the tooth-surrounding tissues. Infection is the etiological cause of the disease, but its extent and severity depend on the immune-inflammatory response of the host. Immune cells use reactive oxygen species to suppress infections, and there is homeostasis between oxidative and antioxidant mechanisms during periodontal health. During periodontitis, however, increased oxidative stress triggers tissue damage, either directly by activating apoptosis and DNA damage or indirectly by activating proteolytic cascades. Periodontal treatment aims to maintain an infection and inflammation-free zone and, in some cases, regenerate lost tissues. Although mechanical disruption of the oral biofilm is an indispensable part of periodontal treatment, adjunctive measures, such as antibiotics or anti-inflammatory medications, are also frequently used, especially in patients with suppressed immune responses. Recent studies have shown that probiotics activate antioxidant mechanisms and can suppress extensive oxidative stress via their ability to activate nuclear factor erythroid 2-related factor 2 (Nrf2). The aim of this narrative review is to describe the essential role of Nrf2 in the maintenance of periodontal health and to propose possible mechanisms to restore the impaired Nrf2 response in periodontitis, with the aid of probiotic and postbiotics.

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. METHODS: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. RESULTS: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing transforming growth factor-β-activated kinase 1 (TAK1) and reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). CONCLUSIONS: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases.

The human (h) transporter hZIP4 is the primary Zn2+ importer in the intestine. hZIP4 is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the Zn2+ deficiency disease acrodermatitis enteropathica (AE). AE can disrupt digestive and immune system homeostasis. A limited number of hZIP4 expression strategies have hindered increasing knowledge about this essential transmembrane protein. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. Both a wild-type and a mutant S. cerevisiae strain, in which the endogenous Zn2+ transporters were deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane-domain-only (mhZIP4-GFP) protein were observed to be present in the plasma membrane in yeast.

Ectopic excitability in pulmonary veins (PVs) is the major cause of atrial fibrillation. We previously reported that the inositol trisphosphate receptor in rat PV cardiomyocytes cooperates with the Na+-Ca2+ exchanger to provoke ectopic automaticity in response to norepinephrine. Here, we focused on adenylyl cyclase (AC) as another effector of norepinephrine stimulation. RT-PCR, immunohistochemistry, and Western blotting revealed that the abundant expression of Ca2+-stimulable AC3 was restricted to the supraventricular area, including the PVs. All the other AC isotypes hardly displayed any region-specific expressions. Immunostaining of isolated cardiomyocytes showed an enriched expression of AC3 along the t-tubules in PV myocytes. The cAMP-dependent response of L-type Ca2+ currents in the PV and LA cells is strengthened by the 0.1 mM intracellular Ca2+ condition, unlike in the ventricular cells. The norepinephrine-induced automaticity of PV cardiomyocytes was reversibly suppressed by 100 µM SQ22536, an adenine-like AC inhibitor. These findings suggest that the specific expression of AC3 along t-tubules may contribute to arrhythmogenic automaticity in rat PV cardiomyocytes.

Peroxisome proliferator-activator receptors (PPARs) regulate lipid and glucose metabolism, control inflammatory processes, and modulate several brain functions. Three PPAR isoforms have been identified, PPARα, PPARβ/δ, and PPARγ, which are expressed in different tissues and cell types. Hereinafter, we focus on PPARα involvement in the pathophysiology of neuropsychiatric and neurodegenerative disorders, which is underscored by PPARα localization in neuronal circuits involved in emotion modulation and stress response, and its role in neurodevelopment and neuroinflammation. A multiplicity of downstream pathways modulated by PPARα activation, including glutamatergic neurotransmission, upregulation of brain-derived neurotrophic factor, and neurosteroidogenic effects, encompass mechanisms underlying behavioral regulation. Modulation of dopamine neuronal firing in the ventral tegmental area likely contributes to PPARα effects in depression, anhedonia, and autism spectrum disorder (ASD). Based on robust preclinical evidence and the initial results of clinical studies, future clinical trials should assess the efficacy of PPARα agonists in the treatment of mood and neurodevelopmental disorders, such as depression, schizophrenia, and ASD.

Oral submucous fibrosis (OSF) is a potentially malignant condition of the oral cavity characterized by progressive fibrosis of the submucosal tissues. OSF is typically associated with the use of betel quid (BQ), a chewing package made of natural products (e.g., areca nut, betel leaves), with or without smokeless tobacco. BQ ingredients contain pro-carcinogenic bioactive compounds, but also potentially protective biomolecules, and we have shown recently that the chemical properties of different BQ recipes vary, which may explain the unequal prevalence of OSF and oral cancer in BQ users in different geographical regions. Hence, this scoping review was aimed at evaluating the existing literature regarding different BQ compounds and their association with OSF. The repository of the National Library of Medicine (MEDLINE/PubMed), medRxiv databases, Google scholar, Baidu scholar, CNKI, and EBSCO were used to search for publications that investigated the association between BQ chewing and OSF up to November 2021. The search terminology was constructed using the keywords "betel quid" and "oral submucous fibrosis", and their associated terms, with the use of Boolean operators. The search was conducted under Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, together with clear inclusion and exclusion criteria. The review showed that the risk of developing OSF varied between different BQ recipes, and that chewing BQ mixtures containing betel inflorescence (BI) significantly increased the risk of OSF, as did the addition of tobacco. Conversely, the use of betel leaf in the mixture was likely to be protective, which may be due to the presence of polyphenols. Although further research is needed to determine the effect of individual BQ ingredients in the development of OSF, our pilot results provide the scope and rationale for informing future chemopreventive strategies for OSF and oral cancer in BQ chewers.

The rate of chronic kidney disease (CKD) is increasing globally, and it is caused by continuous damage to kidney tissue. With time the renal damage becomes irreversible, leading to CKD development. In females, post-menopause lack of estrogen supply has been described as a risk factor for CKD development, and studies targeting post-menopause CKD are scarce. In the present study, we used exosomes isolated from bone marrow mesenchymal stem/stromal cells (BM-MSCs) to test their therapeutic potential against the development of CKD. At first, the menopause model was achieved by surgical bilateral ovariectomy in female albino rats. After that, 100 µg of exosomes was given to ovariectomized rats, and the study continued for 2 months. Changes in urine volume, urine protein content, kidney function biochemical parameters (creatinine and BUN), kidney antioxidant parameters (SOD, GPx and CAT), histological changes, immunohistochemical levels of caspase 3, and the gene expression of NGAL (related to kidney damage), TGFβ1 and αSMA (related to fibrosis and EMT), and caspase 3 (related to apoptosis) were studied. After the ovariectomy, the occurrence of CKD was confirmed in the rats by the drastic reduction of serum estrogen and progesterone levels, reduced urine output, increased urinary protein excretion, elevated serum creatinine and BUN, reduced GPx SOD, and CAT in kidney tissue, degenerative and fibrotic lesions in the histopathological examination, higher immunohistochemical expression of caspase 3 and increased expression of all studied genes. After exosomes administration, the entire chronic inflammatory picture in the kidney was corrected, and a near-normal kidney structure and function were attained. This study shows for the first time that BM-MSCs exosomes are potent for reducing apoptosis and fibrosis levels and, thus, can reduce the chronic damage of the kidneys in females that are in their menopause period. Therefore, MSCs-derived exosomes should be considered a valuable therapy for preserving postmenopausal kidney structure and function and, subsequently, could improve the quality of females' life during menopause.

The purpose of this study was to analyze the regenerative capacity of mesenchymal stem cells (MSCs) in the treatment of fractures. MSCs extracted from patients with osteoporotic hip fractures or hip osteoarthritis undergoing hip replacement surgeries were cultured and injected into mice with femoral fracture. Two experimental models were established, one for the systemic administration of MSCs (n = 29) and another one for local administration (n = 30). Fracture consolidation was assessed by micro-CT and histology. The degree of radiological consolidation and corticalization was better with MSCs from osteoporosis than from osteoarthritis, being significant after systemic administration (p = 0.0302 consolidation; p = 0.0243 corticalization). The histological degree of consolidation was also better with MSCs from osteoporosis than from osteoarthritis. Differences in histological scores after systemic infusion were as follows: Allen, p = 0.0278; Huo, p = 0.3471; and Bone Bridge, p = 0.0935. After local administration at the fracture site, differences in histological scores were as follows: Allen, p = 0.0764; Huo, p = 0.0256; and Bone Bridge, p = 0.0012. As osteoporosis and control groups were similar, those differences depended on an inhibitory influence by MSCs from patients with osteoarthritis. In conclusion, we found an unexpected impairment of consolidation induced by MSCs from patients with osteoarthritis. However, MSCs from patients with osteoporosis compared favorably with cells from patients with osteoarthritis. In other words, based on this study and previous studies, MSCs from patients with osteoporosis do not appear to have worse bone-regenerating capabilities than MSCs from non-osteoporotic individuals of similar age.

Inflammation is a complex program of active processes characterized by the well-orchestrated succession of an initiation and a resolution phase aiming to promote homeostasis. When the resolution of inflammation fails, the tissue undergoes an unresolved inflammatory status which, if it remains uncontrolled, can lead to chronic inflammatory disorders due to aggravation of structural damages, development of a fibrous area, and loss of function. Various human conditions show a typical unresolved inflammatory profile. Inflammatory diseases include cancer, neurodegenerative disease, asthma, right heart disease, atherosclerosis, myocardial infarction, or atrial fibrillation. New evidence has started to emerge on the role, including pro-resolution involvement of chemical mediators in the acute phase of inflammation. Although flourishing knowledge is available about the role of specialized pro-resolving mediators in neurodegenerative diseases, atherosclerosis, obesity, or hepatic fibrosis, little is known about their efficacy to combat inflammation-associated arrhythmogenic cardiac disorders. It has been shown that resolvins, including RvD1, RvE1, or Mar1, are bioactive mediators of resolution. Resolvins can stop neutrophil activation and infiltration, stimulate monocytes polarization into anti-inflammatory-M2-macrophages, and activate macrophage phagocytosis of inflammation-debris and neutrophils to promote efferocytosis and clearance. This review aims to discuss the paradigm of failed-resolution mechanisms (FRM) potentially promoting arrhythmogenicity in right heart disease-induced inflammatory status.

Genome-scale metabolic models (GEMs) are effective tools for metabolic engineering and have been widely used to guide cell metabolic regulation. However, the single gene-protein-reaction data type in GEMs limits the understanding of biological complexity. As a result, multiscale models that add constraints or integrate omics data based on GEMs have been developed to more accurately predict phenotype from genotype. This review summarized the recent advances in the development of multiscale GEMs, including multiconstraint, multiomic, and whole-cell models, and outlined machine learning applications in GEM construction. This review focused on the frameworks, toolkits, and algorithms for constructing multiscale GEMs. The challenges and perspectives of multiscale GEM development are also discussed.

As bulk chemicals, diols have wide applications in many fields, such as clothing, biofuels, food, surfactant and cosmetics. The traditional chemical synthesis of diols consumes numerous non-renewable energy resources and leads to environmental pollution. Green biosynthesis has emerged as an alternative method to produce diols. Escherichia coli as an ideal microbial factory has been engineered to biosynthesize diols from carbon sources. Here, we comprehensively summarized the biosynthetic pathways of diols from renewable biomass in E. coli and discussed the metabolic-engineering strategies that could enhance the production of diols, including the optimization of biosynthetic pathways, improvement of cofactor supplementation, and reprogramming of the metabolic network. We then investigated the dynamic regulation by multiple control modules to balance the growth and production, so as to direct carbon sources for diol production. Finally, we proposed the challenges in the diol-biosynthesis process and suggested some potential methods to improve the diol-producing ability of the host.

This prospective pilot study aimed to evaluate the effect of minocycline-HCl ointment (MO), locally delivered as an adjunct to scaling and root planing (SRP), on subgingival microflora. A total of 59 periodontitis patients received SRP as an initial periodontal therapy. In the selected periodontal pockets with probing depths (PD) of 6-9 mm, the sites that exhibited a positive reaction following a bacterial test using an immunochromatographic device were subsequently treated with MO (SRP + MO group, n = 25). No additional treatment was performed at sites showing a negative reaction (SRP group, n = 34). In addition to subgingival plaque sampling, measurement of clinical parameters including PD, clinical attachment level (CAL), bleeding on probing (BOP), plaque index and gingival index (GI) were performed at baseline and 4 weeks after the initial periodontal therapy. The subgingival microflora were assessed by terminal restriction fragment-length polymorphism analysis. Relative to baseline values, the mean scores for PD-, CAL-, BOP-, and GI-sampled sites were significantly decreased post treatment in both groups (p < 0.01). The intra-comparisons showed a significant decrease in the counts of the genera Eubacterium, Parvimonas, Filifactor, Veillonella, Fusobacterium, Porphyromonas, Prevotella, and unknown species in the SRP + MO group (p < 0.05). Inter-comparisons indicated a significant decrease in the genera Veillonella in the SRP + MO group (p = 0.01). Combination therapy of SRP and local MO induced a change in the subgingival microbial community: particularly, the number of Veillonella spp. was markedly reduced.

Introduction: ILDs are a varied group of diffuse parenchymal lung diseases associated with high morbidity and mortality. Current treatments can only slow their progression but not cure the disease. Other treatments such as oxygen therapy can also be used as support. We know very little about the effects of oxygen therapy on patients with ILDs. The aim of this study was to collect data from the literature in order to determine whether oxygen therapy can actually decrease the mortality rate or whether it is only suitable for supportive therapy for patients with ILDs. Methods: We reviewed the literature since 2010 on oxygen therapy during exercise in patients with ILDs. Studies that used cardio-pulmonary tests were excluded. We only reviewed those that used the 6 min walking test (6MWT) or the free walking test. We located 11 relevant articles. Results: All the articles except a Japanese study showed an augmentation in oxyhaemoglobin saturation during exercise when oxygen was supplied. A 2018 study called AmbOx provided important data on the effects of oxygen therapy during daily activities, showing significant improvements in quality of life. Conclusions: This review showed that the literature on the benefits of oxygen therapy in patients with ILDs does not provide sufficient evidence to draft specific guidelines. It was not possible to conclude whether oxygen therapy has an effect on mortality or can only play a supportive role.

Although the discovery of immune checkpoints was hailed as a major breakthrough in cancer therapy, generating a sufficient response to immunotherapy is still limited. Thus, the objective of this exploratory, hypothesis-generating study was to identify potentially novel peripheral biomarkers and discuss the possible predictive relevance of combining scarcely investigated metabolic and hormonal markers with immune subsets. Sixteen markers that differed significantly between responders and non-responders were identified. In a further step, the correlation with progression-free survival (PFS) and false discovery correction (Benjamini and Hochberg) revealed potential predictive roles for the immune subset absolute lymphocyte count (rs = 0.51; p = 0.0224 *), absolute basophil count (rs = 0.43; p = 0.04 *), PD-1+ monocytes (rs = -0.49; p = 0.04 *), hemoglobin (rs = 0.44; p = 0.04 *), metabolic markers LDL (rs = 0.53; p = 0.0224 *), free androgen index (rs = 0.57; p = 0.0224 *) and CRP (rs = -0.46; p = 0.0352 *). The absolute lymphocyte count, LDL and free androgen index were the most significant individual markers, and combining the immune subsets with the metabolic markers into a biomarker ratio enhanced correlation with PFS (rs = -0.74; p ≤ 0.0001 ****). In summary, in addition to well-established markers, we identified PD-1+ monocytes and the free androgen index as potentially novel peripheral markers in the context of immunotherapy. Furthermore, the combination of immune subsets with metabolic and hormonal markers may have the potential to enhance the power of future predictive scores and should, therefore, be investigated further in larger trials.

In silico antibody discovery is emerging as a viable alternative to traditional in vivo and in vitro approaches. Many challenges, however, remain open to enabling the properties of designed antibodies to match those produced by the immune system. A major question concerns the structural features of computer-designed complementarity determining regions (CDRs), including the role of conformational entropy in determining the stability and binding affinity of the designed antibodies. To address this problem, we used enhanced-sampling molecular dynamics simulations to compare the free energy landscapes of single-domain antibodies (sdAbs) designed using structure-based (DesAb-HSA-D3) and sequence-based approaches (DesAbO), with that of a nanobody derived from llama immunization (Nb10). Our results indicate that the CDR3 of DesAbO is more conformationally heterogeneous than those of both DesAb-HSA-D3 and Nb10, and the CDR3 of DesAb-HSA-D3 is slightly more dynamic than that of Nb10, which is the original scaffold used for the design of DesAb-HSA-D3. These differences underline the challenges in the rational design of antibodies by revealing the presence of conformational substates likely to have different binding properties and to generate a high entropic cost upon binding.

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT's functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT's ability to induce IFN β in response to dsRNA and the basal expression of IFN β and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN β and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA.

Disruption of cerebral iron regulation appears to have a role in aging and in the pathogenesis of various neurodegenerative disorders. Possible unfavorable impacts of iron accumulation include reactive oxygen species generation, induction of ferroptosis, and acceleration of inflammatory changes. Whole-brain iron-sensitive magnetic resonance imaging (MRI) techniques allow the examination of macroscopic patterns of brain iron deposits in vivo, while modern analytical methods ex vivo enable the determination of metal-specific content inside individual cell-types, sometimes also within specific cellular compartments. The present review summarizes the whole brain, cellular, and subcellular patterns of iron accumulation in neurodegenerative diseases of genetic and sporadic origin. We also provide an update on mechanisms, biomarkers, and effects of brain iron accumulation in these disorders, focusing on recent publications. In Parkinson's disease, Friedreich's disease, and several disorders within the neurodegeneration with brain iron accumulation group, there is a focal siderosis, typically in regions with the most pronounced neuropathological changes. The second group of disorders including multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis shows iron accumulation in the globus pallidus, caudate, and putamen, and in specific cortical regions. Yet, other disorders such as aceruloplasminemia, neuroferritinopathy, or Wilson disease manifest with diffuse iron accumulation in the deep gray matter in a pattern comparable to or even more extensive than that observed during normal aging. On the microscopic level, brain iron deposits are present mostly in dystrophic microglia variably accompanied by iron-laden macrophages and in astrocytes, implicating a role of inflammatory changes and blood-brain barrier disturbance in iron accumulation. Options and potential benefits of iron reducing strategies in neurodegeneration are discussed. Future research investigating whether genetic predispositions play a role in brain Fe accumulation is necessary. If confirmed, the prevention of further brain Fe uptake in individuals at risk may be key for preventing neurodegenerative disorders.

The aim of our study was to evaluate the importance of insulin-like growth-factor-binding protein 7 (IGFBP-7) as a potential marker of symptomatic peripheral artery disease (PAD) occurrence. The study group consisted of 145 patients with diagnosed PAD, who qualified for the invasive treatment. The control group consisted of 67 individuals representing the local population and an ischemic heart disease (IHD) group of 88 patients after myocardial infarction or percutaneous coronary intervention. Patients with PAD had significantly higher IGFBP-7 concentrations than control group (1.80 ± 1.62 vs. 1.41 ± 0.45 ng/mL, p = 0.04). No significant differences between PAD patients and IHD patients were found (1.80 ± 1.62 vs. 1.76 ± 1.04 ng/mL, p = 0.783). Patients with multilevel PAD presented significantly higher IGFBP-7 concentrations than patients with aortoiliac PAD-median 1.18 (IQR 0.48-2.23) vs. 1.42 ng/mL (0.71-2.63), p = 0.035. In the group of patients who died or had a major adverse cardiovascular event (MACE) during six months of follow-up, a statistically significant higher IGFBP-7 concentration was found (median 2.66 (IQR 1.80-4.93) vs. 1.36 ng/mL (IQR 0.65-2.34), p = 0.004). It seems that IGFBP-7 is elevated in patients with atherosclerotic lesions-regardless of their locations. Further research should be conducted to verify IGFBP-7 usefulness as a predictor of MACE or death.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.

BACKGROUND: Alcohol consumption by adolescents is responsible for a number of adverse health and social outcomes. Despite the well-established effect of alcohol use on the development of alcoholic liver disease, the relationship between the pattern of alcohol consumption and liver fibrosis is still unclear. This study is a follow-up to work on liver damage from alcohol intoxication. The aim of our study was to explore the early effects of alcohol intoxication on liver fibrosis in adolescents. METHODS: The prospective study included 57 adolescents aged 14-17 years admitted to the emergency department (ED) from February 2017 to June 2018 due to acute alcohol intoxication. Serum levels of amino terminal propeptide of type III procollagen (PIIINP), type IV collagen, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were determined by enzyme-linked immunosorbent assays. RESULTS: There were significant differences in MMP-9 (p = 0.02) and TIMP-1 (p = 0.007) levels between the study and control groups. Liver parameters and selected markers of fibrosis were similar in groups in terms of blood alcohol concentrations (BAC). MMP-9 was positively correlated with alanine aminotransferase (ALT) (r = 0.38; p = 0.004) and total bilirubin (r = 0.39; p = 0.004). Positive significant correlations were also found between TIMP-1 and ALT (r = 0.47; p < 0.001), AST (r = 0.29; p = 0.03) and total bilirubin (r = 0.32; p = 0.02). In receiver operating characteristic (ROC) analysis, MMP-9 (AUC = 0.67, p = 0.02) and TIMP-1 (AUC = 0.69, p = 0.003) allowed for the differentiation of patients with and without alcohol intoxication. CONCLUSION: Our results show that even a single episode of alcohol intoxication in adolescents can lead to imbalance in markers of fibrosis.

Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance <30%. We used this method to identify plasma lipids altered due to vitamin B12 deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B12 deficient samples. This method enables rapid screening of a large number of lipid species in a single experiment and would substantially advance our understanding of the role of lipids in biological processes.

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me3 and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me3 and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and β-catenin-both after incubation with Gal-2. The expression of H3K4me3 and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me3 and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me3 and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me3 and H3K9ac. Since Gal-2, as well as H3K4me3 and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target.

Fatty acid metabolism is closely linked to the progression of gastric cancer (GC), a very aggressive and life-threatening tumor. This study examines linked molecules, such as Sterol Regulatory Element-Binding Protein 1 (SREBP1), ATP Citrate Lyase (ACLY), Acetyl-CoA Synthases (ACSs), Acetyl-CoA Carboxylase (ACC), Fatty Acid Synthase (FASN), Stearoyl-CoA Desaturase 1 (SCD1), CD36, Fatty Acid Binding Proteins (FABPs), and Carnitine palmitoyltransferase 1 (CPT1), as well as their latest studies and findings in gastric cancer to unveil its core mechanism. The major enzymes of fatty acid de novo synthesis are ACLY, ACSs, ACC, FASN, and SCD1, while SREBP1 is the upstream molecule of fatty acid anabolism. Fatty acid absorption is mediated by CD36 and FABPs, and fatty acid catabolism is mediated by CPT1. If at all possible, we will discover novel links between fatty acid metabolism and a prospective gastric cancer target.

Non-resolving pancreatic islet inflammation is widely viewed as a contributor to decreases in β-cell mass and function that occur in both Type 1 and Type 2 diabetes. Therefore, strategies aimed at reducing or eliminating pathological inflammation would be useful to protect islet β-cells. Herein, we described the use of 2',4'-dihydroxy-4-methoxydihydrochalcone (DMC2), a bioactive molecule isolated from an ethanolic extract of Artemisia dracunculus L., as a novel anti-inflammatory agent. The ethanolic extract, termed PMI 5011, reduced IL-1β-mediated NF-κB activity. DMC2 retained this ability, indicating this compound as the likely source of anti-inflammatory activity within the overall PMI 5011 extract. We further examined NF-κB activity using promoter-luciferase reporter constructs, Western blots, mRNA abundance, and protein secretion. Specifically, we found that PMI 5011 and DMC2 each reduced the ability of IL-1β to promote increases in the expression of the Ccl2 and Ccl20 genes. These genes encode proteins that promote immune cell recruitment and are secreted by β-cells in response to IL-1β. Phosphorylation of IκBα and the p65 subunit of NF-κB were not reduced by either PMI 5011 or DMC2; however, phosphorylation of p38 MAPK was blunted in the presence of DMC2. Finally, we observed that while PMI 5011 impaired glucose-stimulated insulin secretion, insulin output was preserved in the presence of DMC2. In conclusion, PMI 5011 and DMC2 reduced inflammation, but only DMC2 did so with the preservation of glucose-stimulated insulin secretion.

We report on the fabrication of single-electron devices based on horse-spleen ferritin particles. At low temperatures the current vs. voltage characteristics are stable, enabling the acquisition of reproducible data that establishes the Coulomb blockade as the main transport mechanism through them. Excellent agreement between the experimental data and the Coulomb blockade theory is demonstrated. Single-electron charge transport in ferritin, thus, establishes a route for further characterization of their, e.g., magnetic, properties down to the single-particle level, with prospects for electronic and medical applications.

Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design.

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated-mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated-mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator's ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.

The tumor microenvironment (TME) plays a key role in promoting and sustaining cancer growth. Adipose tissue (AT), due to its anatomical distribution, is a prevalent component of TME, and contributes to cancer development and progression. Cancer-associated adipocytes (CAAs), reprogrammed by cancer stem cells (CSCs), drive cancer progression by releasing metabolites and inflammatory adipokines. In this review, we highlight the mechanisms underlying the bidirectional crosstalk among CAAs, CSCs, and stromal cells. Moreover, we focus on the recent advances in the therapeutic targeting of adipocyte-released factors as an innovative strategy to counteract cancer progression.

In order to identify factors involved in transcription of human snRNA genes and 3' end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2-and Pcf11 and Ssu72, which are also polyadenylation factors-are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6.

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1 α1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g., α-smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target.

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them.

Ecto-5'-nucleotidase (CD73), the ectoenzyme that together with CD39 is responsible for extracellular ATP hydrolysis and adenosine accumulation, regulates immune/inflammatory processes by controlling innate and acquired immunity cell functions. We previously demonstrated that CD73 is required for the assessment of a controlled allergic sensitization, in mice. Here, we evaluated the response to aerosolized allergen of female-sensitized mice lacking CD73 in comparison with their wild type counterpart. Results obtained show, in mice lacking CD73, the absence of airway hyperreactivity in response to an allergen challenge, paralleled by reduced airway CD23+B cells and IL4+T cells pulmonary accumulation together with reduced mast cells accumulation and degranulation. Our findings indicate CD73 as a potential therapeutic target for allergic asthma.

Neurodegenerative disorders including Parkinson's disease (PD), Huntington's disease (HD) and the most frequent, Alzheimer's disease (AD), represent one of the most urgent medical needs worldwide. Despite a significantly developed understanding of disease development and pathology, treatments that stop AD progression are not yet available. The recent approval of sodium oligomannate (GV-971) for AD treatment in China emphasized the potential value of natural products for the treatment of neurodegenerative disorders. Many current clinical studies include the administration of a natural compound as a single and combination treatment. The most prominent mechanisms of action are anti-inflammatory and anti-oxidative activities, thus preserving cellular survival. Here, we review current natural products that are either approved or are in testing for a treatment of neurodegeneration in AD. In addition to the most important compounds of plant origin, we also put special emphasis on compounds from algae, given their neuroprotective activity and their underlying mechanisms of neuroprotection.

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies.

Erratum for Biomolecules. 2022 Feb 03;12(2):

The HIV-1 capsid (CA) protein has emerged as an attractive therapeutic target. However, all inhibitor designs and structural analyses for this essential HIV-1 protein have focused on the clade B HIV-1 (NL4-3) variant. This study creates, overproduces, purifies, and characterizes the CA proteins from clade A1, A2, B, C, and D isolates. These new CA constructs represent novel reagents that can be used in future CA-targeted inhibitor design and to investigate CA proteins' structural and biochemical properties from genetically diverse HIV-1 subtypes. Moreover, we used surface plasmon resonance (SPR) spectrometry and computational modeling to examine inter-clade differences in CA assembly and binding of PF-74, CPSF-6, and NUP-153. Interestingly, we found that HIV-1 CA from clade A1 does not bind to NUP-153, suggesting that the import of CA core structures through the nuclear pore complex may be altered for viruses from this clade. Overall, we have demonstrated that in silico generated models of the HIV-1 CA protein from clades other than the prototypically used clade B have utility in understanding and predicting biology and antiviral drug design and mechanism of action.

The Gram-negative bacterium Helicobacter pylori colonizes c.a. 50% of human stomachs worldwide and is the major risk factor for gastric adenocarcinoma. Its high genetic variability makes it difficult to identify biomarkers of early stages of infection that can reliably predict its outcome. Moreover, the increasing antibiotic resistance found in H. pylori defies therapy, constituting a major human health problem. Here, we review H. pylori virulence factors and genes involved in antibiotic resistance, as well as the technologies currently used for their detection. Furthermore, we show that next generation sequencing may lead to faster characterization of virulence factors and prediction of the antibiotic resistance profile, thus contributing to personalized treatment and management of H. pylori-associated infections. With this new approach, more and permanent data will be generated at a lower cost, opening the future to new applications for H. pylori biomarker identification and antibiotic resistance prediction.

Tea (Camellia sinensis L.), an important economic crop, is recalcitrant to Agrobacterium-mediated transformation (AMT), which has seriously hindered the progress of molecular research on this species. The mechanisms leading to low efficiency of AMT in tea plants, related to the morphology, growth, and gene expression of Agrobacterium tumefaciens during tea-leaf explant infection, were compared to AMT of Nicotiana benthamiana leaves in the present work. Scanning electron microscopy (SEM) images showed that tea leaves induced significant morphological aberrations on bacterial cells and affected pathogen-plant attachment, the initial step of a successful AMT. RNA sequencing and transcriptomic analysis on Agrobacterium at 0, 3 and 4 days after leaf post-inoculation resulted in 762, 1923 and 1656 differentially expressed genes (DEGs) between the tea group and the tobacco group, respectively. The expressions of genes involved in bacterial fundamental metabolic processes, ATP-binding cassette (ABC) transporters, two-component systems (TCSs), secretion systems, and quorum sensing (QS) systems were severely affected in response to the tea-leaf phylloplane. Collectively, these results suggest that compounds in tea leaves, especially gamma-aminobutyrate (GABA) and catechins, interfered with plant-pathogen attachment, essential minerals (iron and potassium) acquisition, and quorum quenching (QQ) induction, which may have been major contributing factors to hinder AMT efficiency of the tea plant.

Gut microbes have been recognized to convert human bile acids by deconjugation, dehydroxylation, dehydrogenation, and epimerization of the cholesterol core, but the ability to re-conjugate them with amino acids as an additional conversion has been recently described. These new bile acids are known as microbially conjugated bile acids (MCBAs). The aim of this study was to evaluate the MCBAs diversity produced by the gut microbiota through a metabolomics approach. In this study, fresh fecal samples from healthy donors were evaluated to explore the re-conjugation of chenodeoxycholic and 3-oxo-chenodeoxycholic acids by the human gut microbiota. No significant differences were found between the conversion trend of both BAs incubations. The in vitro results showed a clear trend to first accumulate the epimer isoursochenodeoxycholic acid and the dehydroxylated lithocholic acid derivatives in samples incubated with chenodeoxycholic and 3-oxo-chenodeoxycholic acid. They also showed a strong trend for the production of microbially conjugated dehydroxylated bile acids instead of chenodeoxycholic backbone conjugates. Different molecules and isomers of MCBAs were identified, and the new ones, valolithocholate ester and leucolithocholate ester, were identified and confirmed by MS/MS. These results document the gut microbiota's capability to produce esters of MCBAs on hydroxyls of the sterol backbone in addition to amides at the C24 acyl site. This study opens a new perspective to study the BAs diversity produced by the human gut microbiota.

Coronavirus disease 2019 (COVID-19) is still an active global public health issue. Although vaccines and therapeutic options are available, some patients experience severe conditions and need critical care support. Hence, identifying key genes or proteins involved in immune-related severe COVID-19 is necessary to find or develop the targeted therapies. This study proposed a novel construction of an immune-related protein interaction network (IPIN) in severe cases with the use of a network diffusion technique on a human interactome network and transcriptomic data. Enrichment analysis revealed that the IPIN was mainly associated with antiviral, innate immune, apoptosis, cell division, and cell cycle regulation signaling pathways. Twenty-three proteins were identified as key proteins to find associated drugs. Finally, poly (I:C), mitomycin C, decitabine, gemcitabine, hydroxyurea, tamoxifen, and curcumin were the potential drugs interacting with the key proteins to heal severe COVID-19. In conclusion, IPIN can be a good representative network for the immune system that integrates the protein interaction network and transcriptomic data. Thus, the key proteins and target drugs in IPIN help to find a new treatment with the use of existing drugs to treat the disease apart from vaccination and conventional antiviral therapy.

Flavonoids are a diverse group of secondary plant metabolites that play an important role in the regulation of plant development and protection against stressors. The biosynthesis of flavonoids occurs through the activity of several enzymes, including chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H). A functional divergence between some copies of the structural TaCHI and TaF3H genes was previously shown in the allohexaploid bread wheat Triticum aestivum L. (BBAADD genome). We hypothesized that the specific nature of TaCHI and TaF3H expression may be induced by the methylation of the promoter. It was found that the predicted position of CpG islands in the promoter regions of the analyzed genes and the actual location of methylation sites did not match. We found for the first time that differences in the methylation status could affect the expression of TaCHI copies, but not the expression of TaF3Hs. At the same time, we revealed significant differences in the structure of the promoters of only the TaF3H genes, while the TaCHI promoters were highly homologous. We assume that the promoter structure in TaF3Hs primarily affects the change in the nature of gene expression. The data obtained are important for understanding the mechanisms that regulate the synthesis of flavonoids in allopolyploid wheat and show that differences in the structure of promoters have a key effect on gene expression.

Phosphorylation facilitates the regulation of all fundamental biological processes, which has triggered extensive research of protein kinases and their roles in human health and disease. In addition to their phosphotransferase activity, certain kinases have evolved to adopt additional catalytic functions, while others have completely lost all catalytic activity. We searched the Universal Protein Resource Knowledgebase (UniProtKB) database for bifunctional protein kinases and focused on kinases that are critical for bacterial and human cellular homeostasis. These kinases engage in diverse functional roles, ranging from environmental sensing and metabolic regulation to immune-host defense and cell cycle control. Herein, we describe their dual catalytic activities and how they contribute to disease pathogenesis.

Flory's random coil model assumes that conformational fluctuations of amino acid residues in unfolded poly(oligo)peptides and proteins are uncorrelated (isolated pair hypothesis, IPH). This implies that conformational energies, entropies and solvation free energies are all additive. Nearly 25 years ago, analyses of coil libraries cast some doubt on this notion, in that they revealed that aromatic, but also β-branched side chains, could change the 3J(HNHCα) coupling of their neighbors. Since then, multiple bioinformatical, computational and experimental studies have revealed that conformational propensities of amino acids in unfolded peptides and proteins depend on their nearest neighbors. We used recently reported and newly obtained Ramachandran plots of tetra- and pentapeptides with non-terminal homo- and heterosequences of amino acid residues to quantitatively determine nearest neighbor coupling between them with a Ising type model. Results reveal that, depending on the choice of amino acid residue pairs, nearest neighbor interactions either stabilize or destabilize pairs of polyproline II and β-strand conformations. This leads to a redistribution of population between these conformations and a reduction in conformational entropy. Interactions between residues in polyproline II and turn(helix)-forming conformations seem to be cooperative in most cases, but the respective interaction parameters are subject to large statistical errors.

Much has been written about matrix metalloproteinases (MMPs) in health and disease conditions, but their roles in the setting of COVID-19 and associated illnesses remain understudied [...].

MicroRNAs are post-transcriptional regulators of gene expression, and disturbances of their expression are the basis of many pathological states, including cancers. The miRNA pattern in the context of tumor microenvironment explains mechanisms related to cancer progression and provides a potential target of modern therapies. Here we show the miRNA pattern in renal cancer focusing on hypoxia as a characteristic feature of the tumor microenvironment and dysregulation of PTEN, being a major tumor suppressor. Methods comprised the CRSPR/Cas9 mediated PTEN knockout in the Renca kidney cancer cell line and global miRNA expression analysis in both in vivo and in vitro (in normoxic and hypoxic conditions). The results were validated on human cancer models with distinct PTEN status. The increase in miR-210-3p in hypoxia was universal; however, the hypoxia-induced decrease in PTEN was associated with an increase in miR-221-3p, the loss of PTEN affected the response to hypoxia differently by decreasing miR-10b-5p and increasing miR-206-3p. In turn, the complete loss of PTEN induces miR-155-5p, miR-100-5p. Upregulation of miR-342-3p in knockout PTEN occurred in the context of the whole tumor microenvironment. Thus, effective identification of miRNA patterns in cancers must consider the specificity of the tumor microenvironment together with the mutations of key suppressors.

The therapy of depression is challenging and still unsatisfactory despite the presence of many antidepressant drugs on the market. Consequently, there is a continuous need to search for new, safer, and more effective antidepressant therapeutics. Previous studies have suggested a potential association of brain histaminergic/serotoninergic signaling and antidepressant- and anxiolytic-like effects. Here, we evaluated the in vivo antidepressant- and anxiolytic-like effects of the newly developed multiple-active ligand ST-2300. ST-2300 was developed from 5-HT2A/2C inverse agonist pimavanserin (PIM, ACP-103) and incorporates a histamine H3 receptor (H3R) antagonist pharmacophore. Despite its parent compound, ST-2300 showed only moderate serotonin 5-HT2A antagonist/inverse agonist affinity (Ki value of 1302 nM), but excellent H3R affinity (Ki value of 14 nM). In vivo effects were examined using forced swim test (FST), tail suspension test (TST), and the open field test (OFT) in C57BL/6 mice. Unlike PIM, ST-2300 significantly increased the anxiolytic-like effects in OFT without altering general motor activity. In FST and TST, ST-2300 was able to reduce immobility time similar to fluoxetine (FLX), a recognized antidepressant drug. Importantly, pretreatment with the CNS-penetrant H3R agonist (R)-α-methylhistamine reversed the antidepressant-like effects of ST-2300 in FST and TST, but failed to reverse the ST-2300-provided anxiolytic effects in OFT. Present findings reveal critical structural features that are useful in a rational multiple-pharmacological approach to target H3R/5-HT2A/5-HT2C.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in several mechanisms, and the alteration of IL-6 signaling leads to the overactivation of various processes including immunity, inflammation, and hemostasis. Although IL-6 increase has been documented in venous thromboembolic diseases, the exact involvement of IL-6 signaling in deep vein thrombosis (DVT) has not been fully understood. Consequently, we investigated the involvement of IL-6 trans-signaling in inflammatory events occurring in DVT, focusing on the role of the interleukin-6 receptor (IL6-R) Asp358Ala variant. The circulating levels of IL-6, soluble IL6-R (sIL6-R), and soluble glycoprotein 130, as well as the Asp358Ala genotyping, were assessed in a consecutive cohort of DVT patients and healthy controls. The results indicated that IL-6 was higher in DVT compared to controls. Moreover, sIL6-R levels were strongly correlated to Asp358Ala variant in both groups, showing a high frequency of this mutation across all samples. Interestingly, our results showed a high frequency of both Asp358Ala mutation and raised IL-6 levels in DVT patients (OR = 21.32; p ≤ 0.01), highlighting that this mutation could explain the association between IL-6 overactivation and DVT outcome. Overall, this study represents a proof of concept for the targeting of IL-6 trans-signaling as a new strategy for the DVT adjuvant therapy.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80-90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.