The mechanisms underlying late-onset preeclampsia (LOPE) remain unknown. Metabolic disturbances have been implicated as a primary factor in LOPE development. Lipids have been shown to have great clinical value in recent years. This study aimed to use lipidomics to provide evidence for the etiology and potential therapeutic approaches for LOPE. Twenty patients with LOPE and 20 healthy controls were enrolled in this study. Placental lipidomic data were acquired using liquid chromatographymass spectrometry (LC-MS/MS), and the data were analyzed by weighted gene correlation network analysis (WGCNA) and statistical methods. Of 1508 identified lipids, 226 were differentially expressed between the LOPE and control groups. In the LOPE group, the abundance of most unsaturated triglycerides (TG) increased, whereas that of other lipids, including phosphatidylcholine (PC), sphingomyelin, and phosphatidylserine (PS) increased. The WGCNA implied that the correlation network module of lipids was highly related to clinical traits. Pathway analysis revealed that these dysregulated lipids are closely related to glycerophospholipid metabolism. Lipidomics may help identify the pathogenesis underlying placental dysfunction in LOPE patients and provide potential therapeutic targets in the future.

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 μM and 687 μmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 °C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 °C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.

Leishmaniasis is a tropical parasitic disease caused by Leishmania spp. They cause several presentations of illness ranging from cutaneous leishmaniasis to visceral leishmaniasis. The current arsenal of drugs to treat leishmaniasis is limited, and drug resistance further impedes the problem. Therefore, it is necessary to revisit the available information to identify an alternative or new target for treatment. The glycoprotein 63 (gp63), is a potential anti-leishmanial target that plays a significant role in host-pathogen interaction and virulence. Many studies are ongoing to develop gp63 inhibitors or use it as a vaccine target. In this review, we will discuss the potential of gp63 as a drug target. This review summarises the studies focusing on gp63 as a drug target and its inhibitors identified using in silico approaches.

The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 3'-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 105 pM, with detection limits as low as 88 fM under optimal experimental conditions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.

Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the genomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quadruplexes followed by a detailed description of the biophysical and biochemical methods used to characterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs.

The effects of non-enzymatic glycation on the structural and functional properties of human angiogenin (hAng) have been investigated with respect to the formation of advanced glycated end products (AGEs), on prolonged treatment with d-Glucose, d-Fructose and d-Ribose at 37 °C. Fluorescence studies show the formation of fluorescent AGEs which exhibit emission maxima at 406 nm and 435 nm. Glycation of hAng with ribose leads to the maximum loss of its functional characteristic properties, as compared to fructose and glucose, along with the formation of higher oligomers. An increase in the incubation time results in the formation of higher oligomers with a concomitant decrease in the ribonucleolytic activity. The increase in the hydrodynamic radii of the glycated samples compared to native hAng is indicative of structural perturbations. The ribonucleolytic activity and the DNA binding ability of glycated hAng has been investigated by an agarose gel-based assay. Glycated hAng was unable to bind with human placental ribonuclease inhibitor (hRI), otherwise known to form one of the strongest protein-protein interaction systems with an affinity in the femtomolar range.

Poor lifestyle choices and genetic predisposition are factors that increase the number of cancer cases, one example being breast cancer, the third most diagnosed type of malignancy. Currently, there is a demand for the development of new strategies to ensure early detection and treatment options that could contribute to the complete remission of breast tumors, which could lead to increased overall survival rates. In this context, the glycans observed at the surface of cancer cells are presented as efficient tumor cell markers. These carbohydrate structures can be recognized by lectins which can act as decoders of the glycocode. The application of plant lectins as tools for diagnosis/treatment of breast cancer encompasses the detection and sorting of glycans found in healthy and malignant cells. Here, we present an overview of the most recent studies in this field, demonstrating the potential of lectins as: mapping agents to detect differentially expressed glycans in breast cancer, as histochemistry/cytochemistry analysis agents, in lectin arrays, immobilized in chromatographic matrices, in drug delivery, and as biosensing agents. In addition, we describe lectins that present antiproliferative effects by themselves and/or in conjunction with other drugs in a synergistic effect.

Leishmania parasites have an oxidative and chemical defense mechanism called trypanothione system (T[SH]2), the most abundant thiol system in trypanosomatids. This system has a central role in processing pentavalent antimony and resistance has been related to a better capacity to metabolize it through the activation of T[SH]2 enzymatic cascade. A biochemical approach was applied to assess the effect of trivalent (SbIII) and pentavalent antimony (SbV) on Trypanothione Reductase (TR) activity of two Leishmania (Viannia) braziliensis clinical isolates, which were labeled as responder (R) and non-responder (NR) after patient treatment with Glucantime®. Both isolates were characterized based on in vitro susceptibility to SbIII and SbV and trypanothione reductase (TR) activity. SbIII and SbV discriminated susceptibility profiles in all parasite forms, since isolate NR had significantly higher EC50 values than isolate R. Differences were observed in TR activity between promastigotes, axenic amastigotes and intracellular amastigotes: R (0.439 ± 0.009, 0.103 ± 0.01 and 0.185 ± 0.01AU.min-1.μg of protein-1) and NR (1.083 ± 0.04, 0.914 ± 0.04 and 0.343 ± 0.04 AU. min-1.μg of protein-1), respectively. Incubation with SbIII and SbV using each form EC50 value caused a time-dependent differential effect on TR activity suggesting that oxidative defense is related to the antimony susceptibility phenotype. Data gathered here shows a biochemical approach able to discriminate two L. (V.) braziliensis clinical isolates measurements TR activity of promastigotes, axenic amastigotes and intracellular amastigotes.

Natural metabolites present an extraordinary chemo-diversity and have been used as the inspiration for new drugs. Considering the need for new treatments against the neglected parasitic disease leishmaniasis, three semi-synthetic derivatives of natural neolignane licarin A were prepared: O-acetyl (1a), O-allyl (1b), and 5-allyl (1c). Using an ex vivo assay, compounds 1a, 1b, and 1c showed activity against the intracellular amastigotes of Leishmania (L.) infantum, with IC50 values of 9, 13, and 10 μM, respectively. Despite no induction of hemolytic activity, only compound 1b resulted in mammalian cytotoxicity (CC50 = 64 μM). The most potent compounds (1a and 1c) resulted in selectivity indexes >18. The mechanism of action of compound 1c was evaluated by fluorescent/luminescent based techniques and MALDI-TOF/MS. After a short incubation period, increased levels of the cytosolic calcium were observed in the parasites, with alkalinization of the acidocalcisomes. Compound 1c also induced mitochondrial hyperpolarization, resulting in decreased levels of ATP without altering the reactive oxygen species (ROS). Neither plasma membrane damages nor DNA fragmentation were observed after the treatment, but a reduction in the cellular proliferation was detected. Using MALDI-TOF/MS, mass spectral alterations of promastigote proteins were observed when compared to untreated and miltefosine-treated groups. This chemically modified neolignan induced lethal alterations of the bioenergetic and protein metabolism of Leishmania. Future PKPD and animal efficacy studies are needed to optimize this promising natural-derived compound.

The growth differentiation factor 11 (GDF11), a member of the superfamily of the transforming growth factor β, has gained relevance in the last few years due to its remarkable effects in cellular biology, particularly in the nervous system, skeletal muscle, the heart, and many epithelial tissues. Some controversies have been raised about this growth factor. Many of them have been related to technical factors but also the nature of the cellular target. In liver biology and pathobiology, the GDF11 has shown to be related in many molecular aspects, with a significant impact on the physiology and the initiation and progression of the natural history of liver diseases. GDF11 has been involved as a critical regulator in lipid homeostasis, which, as it is well known, is the first step in the progression of liver disease. However, also it has been reported that the GDF11 is involved in fibrosis, senescence, and cancer. Although there are some controversies, much of the literature indicates that GDF11 displays effects tending to solve or mitigate pathological states of the liver, with reasonable evidence of correlation with other organs or systems. To a large extent, the controversy, as mentioned, is due to technical problems, such as the specificity of GDF11 antibodies, confusion with its closer family member, myostatin, and the state of differentiation in the tissues. In the present work, we reviewed the specific effects of GDF11 in the biology and pathobiology of the liver as a potential and promising factor for therapeutic intervention shortly.

Skatole (3-methylindole) is a heterocyclic compound naturally found in the feces of vertebrates and is produced by certain flowers. Skatole has been used in specific products of the perfume industry or as a flavor additive in ice cream. Additionally, skatole is formed by tryptophan pyrolysis of tobacco and has been demonstrated to be a mutagen. Skatole-induced pulmonotoxicity was reliably described in ruminants and rodents, but no studies have been conducted in humans. Initially, we provide basic knowledge and a historical overview of skatole. Then, skatole bacterial formation in the intestine is described, and the importance of the microbiome during this process is evaluated. Increased skatole concentrations could serve as a marker for intestinal disease development. Therefore, the human molecular targets of skatole that may have significant effects on various processes in the human body are described. Ultimately, we suggest a link between skatole intestinal formation in humans and skatole-induced pulmonotoxicity, which should be explored further in the future.

We report herein the synthesis of two non-ionic amphiphiles with a cholesterol hydrophobic moiety that can be used as chemical additives for biochemical studies of membrane proteins. They were designed to show a high similarity with the planar steroid core of cholesterol and small-to-medium polar head groups attached at the C3 position of ring-A on the sterol skeleton. The two Chol-Tris and Chol-DG have a Tris-hydroxymethyl and a branched diglucose polar head group, respectively, which provide them sufficient water solubility when mixed with the "gold standard" detergent n-Dodecyl-β-D-Maltoside (DDM). The colloidal properties of these mixed micelles were investigated by means of surface tension (SFT) measurements and dynamic light scattering (DLS) experiments and showed the formation of globular micelles of about 8 nm in diameter with a critical micellar concentration of 0.20 mM for DDM:Chol-DG and 0.22 mM for DDM:Chol-Tris. We showed that mixed micelles do not alter the extraction potency of a G-protein coupled receptor (GPCR): the human adenosine A2A receptor (A2AR). The thermostabilizing effect of the mixed micelles was confirmed on two GPCRs, A2AR and the growth hormone secretagogue receptor (GHSR). Finally, these two mixed micelles were found suitable for the purification of an active form of A2AR which remained able to bind two ligands of different class i.e. the specific agonist CGS-21680 and the specific inverse agonist ZM-241385. This suggests that Chol-Tris and Chol-DG may be used as a non-ionic alternative to the cholesteryl hemisuccinate (CHS) stabilizing agent.

Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml-1,kcat 1.20 s-1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml-1,kcat 0.23 ml mg-1 s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml-1, kcat 0.88 ml mg-1 s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.

Laboratory mice are typically housed at temperatures below the thermoneutral zone for the species, resulting in cold stress and premature cancellous bone loss. Furthermore, mice are more dependent upon non-shivering thermogenesis to maintain body temperature during spaceflight, suggesting that microgravity-induced bone loss may be due, in part, to altered thermogenesis. Consequently, we assessed whether housing mice at room temperature modifies the skeletal response to simulated microgravity. This possibility was tested using the hindlimb unloading (HLU) model to mechanically unload femora. Humeri were also assessed as they remain weight bearing during HLU. Six-week-old female C57BL6 (B6) mice were housed at room temperature (22 °C) or near thermoneutral (32 °C) and HLU for 2 weeks. Compared to baseline, HLU resulted in cortical bone loss in femur, but the magnitude of reduction was greater in mice housed at 22 °C. Cancellous osteopenia in distal femur (metaphysis and epiphysis) was noted in HLU mice housed at both temperatures. However, bone loss occurred at 22 °C, whereas the bone deficit at 32 °C was due to failure to accrue bone. HLU resulted in cortical and cancellous bone deficits (compared to baseline) in humeri of mice housed at 22 °C. In contrast, fewer osteopenic changes were detected in mice housed at 32 °C. These findings support the hypothesis that environmental temperature alters the skeletal response to HLU in growing female mice in a bone compartment-specific manner. Taken together, species differences in thermoregulation should be taken into consideration when interpreting the skeletal response to simulated microgravity.

Triabin, a lipocalin-like thrombin inhibitor from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, exhibits effective inhibition comparable to hirudin despite binding exclusively at exosite I. Interestingly, it was reported that higher triabin doses would not inhibit thrombin completely, which makes it a promising antithrombotic candidate agent with a larger therapeutic window. However, few structural and functional studies about triabin have been reported in the past three decades, mostly due to the lack of a reliable and practicable recombinant expression technology for this seemingly small protein. In this work, we have adopted the SUMO fusion technology for the expression of triabin in E. coli cells-with facile refolding and purification procedures-and the bioactive triabin was produced in ∼12 mg/L culture medium. Subsequently, the structure-function studies through extensive site-directed mutagenesis reveal that triabin's Phe-106 involved in the hydrophobic contacts plays a surprisingly important role in the thrombin inhibition, in contrast to the negatively charged residues Asp-135 or Glu-128 involved in the salt-bridge interaction. As such, this study complements our understanding of the interaction mechanism of natural thrombin inhibitors, which should facilitate the development of anticoagulant drugs with a novel mode of action against thrombin.

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

The significant resistance to currently available chemotherapeutics makes treatment for TNBC a key clinical concern. Herein, we studied the anti-cancer potentials of synthetic cannabidiol (CBD) and Tetrahydrocannabivarin (THCV) when used alone or in combination with doxorubicin (DOX) against MDA-MB-231 resistant cells. Pre-treatment with CBD and THCV significantly increased the cytotoxicity of DOX in MDA-MB-231 2D and 3D cultures that were DOX-resistant. Transcriptomics and Proteomics studies revealed that CBD and THCV, by downregulating PD-L1, TGF-β, sp1, NLRP3, P38-MAPK, and upregulating AMPK induced apoptosis leading to improved DOX's chemosensitivity against DOX resistant MDA-MB-231 tumors in BALB/c nude mice. CBD/THCV in combination with DOX significantly inhibited H3k4 methylation and H2K5 acetylation as demonstrated by western blotting and RT-PCR. Based on these findings, CBD and THCV appear to counteract histone modifications and their subsequent effects on DOX, resulting in chemo-sensitization against MDA-MB-231 resistant cancers.

Inflammatory bowel disease is a chronic inflammatory disease and has periods of recurrence and remission. Improper immune responses to gut flora bacteria, along with genetic susceptibility, appear to be involved in causing this complex disease. It seems dysbiosis and oxidative stress may also be involved in IBD pathogenesis. A significant number of clinical studies have shown an interesting association between sleep disturbances and IBD. Studies in animal models have also shown that sleep deprivation has a significant effect on the pathogenesis of IBD and can aggravate inflammation. These interesting findings have drawn attention to melatonin, a sleep-related hormone. Melatonin is mainly produced by the pineal gland, but many tissues in the body, including the intestines, can produce it. Melatonin can have an interesting effect on the pathogenesis of IBD. Melatonin can enhance the intestinal mucosal barrier, alter the composition of intestinal bacteria in favor of bacteria with anti-inflammatory properties, regulate the immune response, alleviate inflammation and attenuate oxidative stress. It seems that, melatonin supplementation is effective in relieving inflammation and healing intestinal ulcers in IBD animal models. Some clinical studies have also shown that melatonin supplementation as an adjuvant therapy may be helpful in reducing disease activity in IBD patients. In this review article, in addition to reviewing the effects of sleep disturbances and melatonin on key mechanisms involved in the pathogenesis of IBD, we will review the findings of clinical studies regarding the effects of melatonin supplementation on IBD treatment.

The DNMT3A DNA methyltransferase is an important epigenetic enzyme that is frequently mutated in cancers, particularly in AML. The heterozygous R736H mutation in the FF-interface of the tetrameric enzyme is the second most frequently observed DNMT3A cancer mutation, but its pathogenic mechanism is unclear. We show here that R736H leads to a moderate reduction in catalytic activity of 20-40% depending on the substrate, but no changes in CpG specificity, flanking sequence preferences and subnuclear localization. Strikingly, R736H showed a very strong stimulation by DNMT3L and the R736H/DNMT3L complex was 3-fold more active than WT/DNMT3L. Similarly, formation of mixed R736H/DNMT3A WT FF-interfaces led to an increased activity. R736H/DNMT3L and mixed R736H/DNMT3A WT FF-interfaces were less stable than interfaces not involving R736H, suggesting that an increased flexibility of the mixed interfaces stimulates catalytic activity. Our data suggest that aberrant activity of DNMT3A R736H may lead to DNA hypermethylation in cancer cells which could cause changes in gene expression.

Sertoli cells provide structural and nutritional support for germ cell development. They actively metabolize glucose and convert it into lactate, which is an important source of energy for germ cells. They also oxidize fatty acids (FA), stored as triacylglycerides (TAGs) within lipid droplets (LD), to fulfill their own energy requirements. So, the combined regulation of lactate production and FA metabolism may be relevant to the physiology of seminiferous tubules. Resveratrol (RSV) is a nutritional supplement found primarily in red grape skin that exhibits multiple beneficial health effects: it is cardioprotective, anti-inflammatory, anticancer, and antiaging. The aim of this study was to evaluate the effect of RSV in Sertoli cells lactate production and lipid metabolism. Sertoli cell cultures obtained from 20-day-old rats were incubated for different times with 10 or 50 μM RSV. RSV treatment increased lactate production and glucose consumption. These increments were accompanied by a rise in GLUT1 expression, which is the main glucose transporter in Sertoli cells. On the other hand, RSV decreased LD content and TAG levels. In addition, an increase in ATGL and FAT/CD36 mRNA levels was observed, which suggests augmented cytoplasmatic FA availability. RSV treatment also increased P-ACC levels, which might indicate that RSV promotes FA transport into the mitochondria to be oxidized. An enhanced expression of LCAD and MCAD, enzymes that participate in the oxidation of FA, was also observed. Altogether, these results suggest that RSV simultaneously regulates Sertoli cells lactate production and lipid metabolism, ensuring an adequate energetic balance both in germ and Sertoli cells.

The small heat shock protein (sHsp) family is a group of proteins in which some are induced in response to external stimuli, such as environmental and pathological stresses, while others are constitutively expressed. They show chaperone-like activity, protect cells from apoptosis, and maintain cytoskeletal architecture. Short sequences or fragments ranging from approximately 19-20 residues in sHsps were shown to display chaperone activity in vitro. These sequences are termed sHsp-derived mini-peptides/mini-chaperones. These peptides offer an advantage in providing protective and therapeutic effects over full-length proteins owing to their small molecular weight and easy uptake into the cells. Research on sHsp mini-chaperone therapy has recently received attention and advanced tremendously. sHsp mini-chaperones have shown a wide range of therapeutic effects, such as anti-aggregation of proteins, anti-apoptotic, anti-inflammatory, anti-oxidant, senolytic, and anti-platelet activity. The administration of mini-chaperones into the several disease animal models, including experimental autoimmune encephalomyelitis, cataract, age-related macular degeneration, glaucoma, and thrombosis through various routes reduced symptoms or prevented the progression of the disease. However, it was found that the therapeutic potential of sHsp mini-chaperones is limited by their short turnover and enzymatic degradation in circulation. Nonetheless, carrier molecules approach such as nanoparticles, cell penetration peptides, and extracellular vesicles increased their efficacy by enhancing the uptake, retention time, protection from enzymatic degradation, and site-specific delivery without altering their biological activity. In this context, this review highlights the recent advances in the therapeutic potential of sHsp-derived mini-chaperones, their effect in experimental animal models, and approaches for increasing their efficacy.