The discovery of leptin in the 1990s led to a reconsideration of adipose tissue (AT) as not only a fatty acid storage organ, but also a proper endocrine tissue. AT is indeed capable of secreting bioactive molecules called adipokines for white AT or batokines for brown/beige AT, which allow communication with numerous organs, especially brain, heart, liver, pancreas, and/or the vascular system. Adipokines exert pro or anti-inflammatory activities. An equilibrated balance between these two sets ensures homeostasis of numerous tissues and organs. During the development of obesity, AT remodelling leads to an alteration of its endocrine activity, with increased secretion of pro-inflammatory adipokines relative to the anti-inflammatory ones, as shown in the graphical abstract. Pro-inflammatory adipokines take part in the initiation of local and systemic inflammation during obesity and contribute to comorbidities associated to obesity, as detailed in the present review.

Tuberculosis is one of the most common infectious diseases and has been a major burden for a long time now. Increasing drug resistance in TB is slowing down the process of disease treatment. Mycobacterium tuberculosis, the causative agent of TB is known to have a cascade of virulence factors to fight with host's immune system. The phosphatases (PTPs) of Mtb plays a critical role as these are secretory in nature and help the survival of bacteria in host. Researchers have been trying to synthesize inhibitors against a lot of virulence factors of Mtb but recently the phosphatases have gained a lot of interest due to their secretory nature. This review gives a concise overview of virulence factors of Mtb with emphasis on mPTPs. Here we discuss the current scenario of drug development against mPTPs.

Uveal melanoma (UM) is the primary ocular cancer with upto 50% of patients dying from metastasis. Although rare, it is deadly as patients with metastatic UM seldom survive beyond 18 months after diagnosis. Chemotherapeutics have no proven efficacy, including immunotherapies that have been tried as current treatment options but produce marginal improvement in overall survival for UM patients. While therapeutics are low in efficacy, there is an urgent need to explore novel targets in the treatment of UM. This review provides an update on the contribution of inflammation to UM with a focus on exploring potential therapeutic targets related to the inflammatory tumour microenvironment. As a tumour promoting event, inflammation is one of the hallmarks of cancers. The presence of the inflammatory phenotype characterised by the abundance of immune mediators and proinflammatory cytokines surrounding UM tumours, is a potential area to explore novel therapeutic targets. Despite decades of investigation regarding the role UM tumour microenvironment has played, that of inflammation in UM progression remains poorly understood. With advancement of technologies, an understanding of the prognosis of UM has been accelerated. Excitingly, novel therapeutic targets related to the inflammatory tumour microenvironment have been identified and relevant studies are underway in their preliminary phases, illustrating optimistic results.

Cancer is one of the main diseases currently afflicting mankind, being difficult to treat and generating thousands of deaths per year. As a result, researchers around the world are constantly searching for new therapeutic strategies to increase the survival rate of patients. In this regard, SIRT5 may be a promising therapeutic target due to its involvement in many metabolic pathways. Notably, SIRT5 has a dual role in the context of cancer, being able to act as a tumor suppressor in some types of cancer and behaving as an oncogene in others. Interestingly, the performance of SIRT5 is not specific and is highly dependent on the cellular context. As a tumor suppressor, SIRT5 prevents the Warburg effect, increases protection against ROS and reduces cell proliferation and metastasis, while as an oncogene it has the opposite effects as well as increasing resistance to chemotherapeutics and/or radiation. In this way, the aim of this work was to identify in which cancers SIRT5 has beneficial effects and in which deleterious ones based on their molecular characteristics. Furthermore, it was analyzed whether it is feasible to use this protein as a therapeutic target, either enhancing its activity or inhibiting it as appropriate.

Metabolic disease is a complex disorder defined by a group with interrelated factors. There is growing evidence that obesity can lead to a variety of metabolic diseases, including diabetes and cardiovascular disease. Excessive adipose tissue (AT) deposition and ectopic accumulation can lead to increased peri-organ AT thickness. Dysregulation of peri-organ (perivascular, perirenal, and epicardial) AT is strongly associated with metabolic disease and its complications. The mechanisms include secretion of cytokines, activation of immunocytes, infiltration of inflammatory cells, involvement of stromal cells, and abnormal miRNA expression. This review discusses the associations and mechanisms by which various types of peri-organ AT affect metabolic diseases while addressing it as a potential future treatment strategy.

The Wnt/β-catenin signaling pathway is a highly conserved pathway that is involved in cell development, proliferation, differentiation, apoptosis and autophagy. Among these processes, apoptosis and autophagy occur physiologically during host defense and the maintenance of intracellular homeostasis. Mounting evidence suggests that the crosstalk between Wnt/β-catenin-regulated apoptosis and autophagy has broad functional significance in various diseases. Herein, we summarize the recent studies in understanding the role of the Wnt/β-catenin signaling pathway in apoptosis and autophagy, and draw the following conclusions: a) For apoptosis, the regulation of Wnt/β-catenin is generally positive. However, a small amount of evidence indicates the presence of a negatively regulated relationship between Wnt/β-catenin and apoptosis; b) Wnt/β-catenin influences the occurrence and development of autophagy by regulating autophagy-related factors, and these factors in turn affect Wnt/β-catenin pathway; c) Wnt/β-catenin always balances the molecular damage caused by the crosstalk between autophagy and apoptosis in a compensatory manner. Understanding the specific role of the Wnt/β-catenin signaling pathway during different stages of autophagy and apoptosis may provide new insights into the progression of related diseases regulated by the Wnt/β-catenin signaling pathway.

As a multi-potency cytokine, leptin not only plays a crucial role in controlling weight and energy homeostasis but also participates in the metabolic balance in the human body. Leptin is a small helical protein with a molecular weight of 16 kDa. It can interact with multiple subtypes of its receptors to initiate intracellular signal transduction and exerts physiological effects. Disturbances in leptin signaling may lead to obesity and a variety of metabolic diseases. Leptin was also found to be a critical factor in many diseases of the elderly. In this review, we focus on recent advances in the structural and molecular mechanisms of leptin signaling through its receptors with the aim of a deeper understanding of leptin-related diseases.

RNase R is a member of the RNA exonuclease family that digests RNA in the 3'-5' direction. Previous studies have identified RNase R from Mycoplasma genitalium (MgR) as the only RNA exonuclease that is sensitive to 2'-O-methylation (Nm) modification. However, the mechanism underlying this characteristic is not well understood. In this study, we aimed to explore the molecular mechanism of RNase R Nm sensitivity using an improved assay that can better evaluate Nm sensitivity. By comparing the sequences of five wild-type RNase R variants from Mycoplasma, we identified the importance of loop 18 in Nm sensitivity. Furthermore, we demonstrated the critical roles of L283, T278, and T279 within loop18. Our findings deepen the understanding of the molecular mechanism of why MgR is sensitive to Nm and provide a potential direction of protein engineering for applications.

Although the mechanisms as well as pathways associated with cancer stem cell (CSC) maintenance, expansion, and tumorigenicity have been extensively studied and the role of tumor cell (TC)-derived exosomes in this process is well understood, there is a paucity of research focusing specifically on the functional mechanisms of CSC-derived exosomes (CSC-Exo)/-exosomal-ncRNAs and their impact on malignancy. This shortcoming needs to be addressed, given that these vesicular and molecular components of CSCs could have a great impact on the cancer initiation, progression, and recurrence through their interaction with other key tumor microenvironment (TME) components, such as MSCs/MSC-Exo and CAFs/CAF-Exo. In particular, understanding CSCs/CSC-Exo and its crosstalk with MSCs/MSC-Exo or CAFs/CAF-Exo that are associated with the proliferation, migration, differentiation, angiogenesis, and metastasis through an enhanced process of self-renewal, chemotherapy as well as radiotherapy resistance may aid cancer treatment. This review contributes to this endeavor by summarizing the characteristic features and functional mechanisms of CSC-Exo/MSC-Exo/CAF-Exo and their mutual impact on cancer progression and therapy resistance.

Gestational diabetes mellitus (GDM) is a metabolic disorder in pregnancy whose prevalence is on the rise. Reports suggest a likely association between inflammation and maternal GDM. A balance between pro and anti-inflammatory cytokines is necessary for the regulation of maternal inflammation system throughout pregnancy. Along with various inflammatory markers, fatty acids also act as pro-inflammatory molecules. However, studies reporting the role of inflammatory markers in GDM are contradictory, suggesting the need of more studies to better understand the role of inflammation in pregnancies complicated by GDM. Inflammatory response can be regulated by angiopoietins suggesting a link between inflammation and angiogenesis. Placental angiogenesis is a normal physiological process which is tightly regulated during pregnancy. Various pro and anti-angiogenic factors influence the regulation of the feto-placental vascular development. Studies evaluating the levels of angiogenic markers in women with GDM are limited and the findings are inconsistent. This review summarizes the available literature on fatty acids, inflammatory markers and angiogenesis in women with GDM. We also discuss the possible link between them and their influence on placental development in GDM.

The merits of stem cell therapy and research are undisputed due to their widespread usage in the treatment of neurodegenerative diseases and demyelinating disorders. Cell replacement therapy especially revolves around stem cells and their induction into different cell lineages both adult and progenitor - belonging to each germ layer, prior to transplantation or disease modeling studies. The nervous system is abundant in glial cells and among these are oligodendrocytes capable of myelinating new-born neurons and remyelination of axons with lost or damaged myelin sheath. But demyelinating diseases generate tremendous deficit between myelin loss and recovery. To compensate for this loss, analyze the defects in remyelination mechanisms as well as to trigger full recovery in such patients mesenchymal stem cells (MSCs) have been induced to transdifferentiate into oligodendrocytes. But such experiments are riddled with problems like prolonged, tenuous and complicated protocols that stretch longer than the time taken for the spread of demyelination-associated after-effects. This review delves into such protocols and the combinations of different molecules and factors that have been recruited to derive bona fide oligodendrocytes from in vitro differentiation of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and MSCs with special focus on MSC-derived oligodendrocytes.

It has been established that translationally controlled tumor protein (TCTP), also called histamine releasing factor (HRF), exhibits cytokine-like activities associated with initiation of allergic responses only after forming dimers (dTCTP). Agents that inhibit dTCTP by preventing its dimerization or otherwise block its function, also block development of allergic reactions, thereby serving as potential drugs to treat allergic diseases. Several lines of evidence have proven that peptides and antibodies that specifically inhibit the interactions between dTCTP and either its putative receptor or immunoglobulins exhibit significant in vivo efficacy as potential anti-inflammatory agents in murine models of allergic inflammatory diseases. This review highlights the development of several inhibitors targeting dTCTP and discusses how they affect the pathophysiological processes of allergic and inflammatory diseases in several animal models and offers new perspectives on anti-allergic drug discovery.

Mice thermoneutral zone lies at temperatures much higher than expected when considering the geographical extension of the species. Growing evidence shows that mouse-dependent thermogenesis experimentation needs to cope with temperatures below those at which the animals are most comfortable. The associated physiological changes interfere with experimental results, thereby highlighting the apparently trivial subject of room-temperature. Working at above 25 °C is difficult for researchers and animal care technicians. Herein, we explore alternative solutions related to living habits of wild mice that could improve translation of research on mice to humans. Standard murine environments are often colder than those in laboratory facilities and their behavior is mainly characterized by a gregarious, nesting and exploratory way of life. Optimization of their thermal environment can thus also be achieved by avoiding individual housing and providing high-quality nesting material and devices that would allow locomotor activity, hence muscle thermogenesis. These options have additional relevance in terms of animal welfare. When precise monitoring of the temperature is required, temperature-controlled cabinets can be used for the duration of the experiments. During the manipulation of mice, a heated laminar flow hood or tray could create an optimized microenvironment. The specification of temperature-related data in publications should contain information on the translatability of the described mouse models to humans. Furthermore, publications should describe the premises of the laboratory in relation to housing possibilities and murine behavior.

The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% β-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until Cm of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 ± 0.87 °C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 ± 15 μM and specific activity at optimum pH of 2.10 ± 0.07 μmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10-15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme that reduces the oxidized methionine (Met-O) in proteins to methionine (Met). Its pivotal role in the cellular processes has been well established by overexpressing, silencing, and knocking down MsrA or deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA in bacterial pathogens. To elucidate this, we infected mouse bone marrow-derived macrophages (BMDMs) with recombinant Mycobacterium smegmatis strain (MSM), secreting a bacterial MsrA or M. smegmatis strain (MSC) carrying only the control vector. BMDMs infected with MSM induced higher levels of ROS and TNF-α than BMDMs infected with MSC. The increased ROS and TNF-α levels in MSM-infected BMDMs correlated with elevated necrotic cell death in this group. Further, RNA-seq transcriptome analysis of BMDMs infected with MSC and MSM revealed differential expression of protein and RNA coding genes, suggesting that bacterial-delivered MsrA could modulate the host cellular processes. Finally, KEGG pathway enrichment analysis identified the down-regulation of cancer-related signaling genes in MSM-infected cells, indicating that MsrA can potentially regulate the development and progression of cancer.

Prokaryotic Argonaute (pAgo) proteins are programmable nucleases with great promise in genetic engineering and biotechnology. Previous studies identified several DNA-targeting pAgo nucleases from mesophilic and thermophilic prokaryotic species that are active in various temperature ranges. However, the effects of temperature on the specificity of target recognition and cleavage by pAgos have not been studied. Here, we describe a thermostable pAgo nuclease from the thermophilic bacterium Thermobrachium celere, TceAgo. We show that TceAgo preferentially uses 5'-phosphorylated small DNA guides and can perform specific cleavage of both single-stranded and double-stranded DNA substrates in a wide range of temperatures. Single-nucleotide mismatches between guide and target molecules differently change the reaction efficiency depending on the mismatch position, with the fidelity of target recognition greatly increased at elevated temperatures. Thus, TceAgo can serve as a tool to allow specific detection and cleavage of DNA targets in a temperature-dependent manner. The results demonstrate that the specificity of programmable nucleases can be strongly affected by the reaction conditions, which should be taken into account when using these nucleases in various in vitro and in vivo applications.

The misfolding and conformational transformation of prion protein (PrP) are crucial to the progression of prion diseases. Screening for available natural inhibitors against prion proteins can contribute to the rational design and development of new anti-prion drugs and therapeutic strategies. The prion neuropeptide, PrP106-126 is commonly used as a model peptide of the abnormal PrPSc, and a number of potential inhibitors were explored against the amyloid fibril formation of PrP106-126. The well-known sesquiterpene lactone, artemisinin, shows diverse biological functions in anti-malarial, anti-cancer and lowering glucose. However, its inhibitory effect on PrP106-126 fibrillation is unclear. In this work, we selected two sesquiterpene lactones, artemisinin (1) and artesunate (2), to explore their roles in PrP106-126 aggregation by a series of physicochemical and biochemical methods. The results demonstrated that 1 and 2 could effectively impede the formation of amyloid fibrils and remodel the preformed fibrils. The binding of the small molecules to PrP106-126 was dominated by electrostatic, hydrophobic and hydrogen bonding interactions. In addition, both compounds exhibited neuroprotective effects by reducing peptide oligomerization. 2 showed better inhibition and regulation on peptide aggregation and cellular viability than 1 due to its specific succinate modification. Our study provides the information of sesquiterpene lactones to prevent PrP fibril formation and other related amyloidosis.

Trichoderma reesei (anamorph Hypocrea jecorina) produces an extracellular beta-galactosidase from Glycoside Hydrolase Family 35 (TrBga1). Hydrolysis of xyloglucan oligosaccharides (XGOs) by TrBga1 has been studied by hydrolysis profile analysis of both tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seed storage xyloglucans using PACE and MALDI-ToF-MS for separation, quantification and identification of the hydrolysis products. The TrBga1 substrate preference for galactosylated oligosaccharides from both the XXXG- and XXXXG-series of jatobá xyloglucan showed that the doubly galactosylated oligosaccharides were the first to be hydrolyzed. Furthermore, the TrBga1 showed more efficient hydrolysis against non-reducing end dexylosylated oligosaccharides (GLXG/GXLG and GLLG). This preference may play a key role in xyloglucan degradation, since galactosyl removal alleviates steric hindrance for other enzymes in the xyloglucanolytic complex resulting in complete xyloglucan mobilization. Indeed, mixtures of TrBga1 with the α-xylosidase from Escherichia coli (YicI), which shows a preference towards non-galactosylated xyloglucan oligosaccharides, reveals efficient depolymerization when either enzyme is applied first. This understanding of the synergistic depolymerization contributes to the knowledge of plant cell wall structure, and reveals possible evolutionary mechanisms directing the preferences of debranching enzymes acting on xyloglucan oligosaccharides.

Oxidative stress is one of the elements causing aging and related diseases. Inhibiting Nrf2 activity or increasing oxidative pressure can replicate the deficits of premature aging. SIRT6 is one of the few proteins that can regulate both life span and aging. Deletion of SIRT6 in human cells impairs the antioxidant capacity of cells, which results in the accumulation of intracellular reactive oxygen species and DNA oxidation products. Characterization of the binding of Nrf2 with SIRT6 is critical for understanding the modulation of Nrf2-correlated cell activities by SIRT6. The yeast two-hybrid experiments showed that the binding of Nrf2 with SIRT6 is mediated by Neh1 and Neh3 domains. The elimination of the Neh1 and Neh3 domains decreased the binding stability and free energy, according to the molecular dynamic analysis. The roles of theses domains in mediating the binding were confirmed by co-immunoprecipitation. In cells transfected with the small interfering RNA (siRNA) targeting the Nrf2 Neh1 domain and plasmids overexpressing domain-mutant Nrf2, it was discovered that Nrf2 lost its activity to stimulate the transcription of antioxidant genes in the absence of Neh1 and Neh3 domains.

Antipsychotic drugs are prescribed to a wide range of individuals to treat mental health conditions including schizophrenia. However, antipsychotic drugs cause bone loss and increase fracture risk. We previously found that the atypical antipsychotic (AA) drug risperidone causes bone loss through multiple pharmacological mechanisms, including activation of the sympathetic nervous system in mice treated with clinically relevant doses. However, bone loss was dependent upon housing temperature, which modulates sympathetic activity. Another AA drug, olanzapine, has substantial metabolic side effects, including weight gain and insulin resistance, but it is unknown whether bone and metabolic outcomes of olanzapine are also dependent upon housing temperature in mice. We therefore treated eight week-old female mice with vehicle or olanzapine for four weeks, housed at either room temperature (23 °C) or thermoneutrality (28-30 °C), which has previously been shown to be positive for bone. Olanzapine caused significant trabecular bone loss (-13% BV/TV), likely through increased RANKL-dependent osteoclast resorption, which was not suppressed by thermoneutral housing. Additionally, olanzapine inhibited cortical bone expansion at thermoneutrality, but did not alter cortical bone expansion at room temperature. Olanzapine also increased markers of thermogenesis within brown and inguinal adipose depots independent of housing temperature. Overall, olanzapine causes trabecular bone loss and inhibits the positive effect of thermoneutral housing on bone. Understanding how housing temperature modulates the impact of AA drugs on bone is important for future pre-clinical studies, as well as for the prescription of AA drugs, particularly to older adults and adolescents who are most vulnerable to the effects on bone.

This study aimed to describe the secretome of mesenchymal stem cells derived from feline adipose tissue (AD-MSCs) and compare the effects of different culture conditions on AD-MSC proteomics using a shotgun approach. Adipose tissue was collected from 5 female cats and prepared to culture. Conditioned media was collected at third passage, in which the cells were cultured under 4 conditions, normoxia with fetal bovine serum (N + FBS), hypoxia with FBS (H + FBS), normoxia without FBS (N - FBS), and hypoxia without FBS (H - FBS). Then, the secretome was concentrated and prepared for proteomic approaches. Secretomes cultured with FBS-free medium had more than twice identified proteins in comparison with the secretomes cultured with FBS. In contrast, hypoxic conditions did not increase protein amount and affected only a small proteome fraction. Relevant proteins were related to the extracellular matrix promoting environmental modulation, influencing cell signaling pathways, and providing a suitable environment for cell proliferation and maintenance. Moreover, other proteins were also related to cell adhesion, migration and morphogenesis. Culture conditions can influence protein abundance in AD-MSC secretome, and can give also more specificity to cell and cell-free treatments for different diseases.

Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is still neither vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation. In the present study, we describe an experimental screening of 2,320 compounds from the chemical library of the Muséum National d'Histoire Naturelle of Paris on ZIKV NS2B-NS3 protease. A total of 96 hits were identified with 90% or more of inhibitory activity at 10 μM. Amongst the most active compounds, five were analyzed for their inhibitory mechanisms by kinetics assays and computational approaches such as molecular docking. 2-(3-methoxyphenoxy) benzoic acid (compound 945) show characteristics of a competitive inhibition (Ki = 0.49 μM) that was corroborated by its molecular docking at the active site of the NS2B-NS3 protease. Taxifolin (compound 2292) behaves as an allosteric inhibitor whereas 3,8,9-trihydroxy-2-methyl-1H-phenalen-1-one (compound 128), harmol (compound 368) and anthrapurpurin (compound 1499) show uncompetitive inhibitions. These new NS2B-NS3 protease inhibitors are valuable hits to further hit-to-lead optimization.

When tumoral cell expansion exceeds the vascular supply, regions of hypoxia or low oxygen concentration are generated promoting the formation of new vessels through cell proliferation and migration. Viral G protein-coupled receptor (vGPCR) is associated to Kaposi's sarcoma pathology and induces a paracrine transformation when is stably expressed in murine endothelial cells activating hypoxia-induced transcription factors. Previously, we reported the antiproliferative actions of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in endothelial cells transformed by the vGPCR (SVEC-vGPCR). Herein, we further investigated if pro-angiogenic factors as AP-1, HIF-1α and VEGF are modulated by 1α,25(OH)2D3. We found by qRT-PCR analysis that the mRNA level of JunB, a negative regulator of cell proliferation, was similarly increased at all-time points tested after 1α,25(OH)2D3 treatment in SVEC-vGPCR cells. Also, mRNA levels of the pro-angiogenic factor c-Fos, which induces tumor invasion, were only decreased during one short period treatment. In addition, Hif-1α mRNA and protein levels were significantly reduced after 1α,25(OH)2D3 treatment in a VDR dependent fashion. However, mRNA levels of the angiogenic activator Vegf, promoted in turn by Hif-1α expression, were surprisingly high depending on VDR expression as well. Moreover, Egr-1, which has been reported to induce VEGF expression independently of HIF-1α, diminished its expression with 1α,25(OH)2D3 treatment, fact that was related to the decline of p-ERK1/2. Altogether, these results suggest a negative modulation of some pro-angiogenic factors like AP-1 and HIF-1α, as part of the antiproliferative mechanism of 1α,25(OH)2D3 in SVEC-vGPCR endothelial cells.

The wood frog, Rana sylvatica, employs freeze tolerance as a winter survival strategy in seasonally cold environments. At subzero temperatures, up to 65-70% of total body water can freeze in extracellular spaces, halting vital functions (breathing, heartbeat) and causing ischemia that, in turn, can have numerous consequences including the generation of damaging reactive oxygen species (ROS). NADPH serves as a key donor of reductive power for most ROS detoxifying enzymes and can be generated by several metabolic pathways. One source of NADPH reducing power is the NADP-dependent isocitrate dehydrogenase (IDH) reaction. The present study evaluated the properties and regulation of IDH from skeletal muscle of R. sylvatica when frogs were exposed to stress conditions: freezing, dehydration or anoxia. Purified IDH exhibited higher affinity for isocitrate under all stress conditions as compared to controls, suggesting that the enzyme is primed to synthesize NADPH relative to the control state. Immunoblotting showed reduced serine and threonine phosphorylation of muscle IDH from frozen frogs and decreased serine phosphorylation on IDH from dehydrated frogs relative to control and anoxic states, demonstrating a reversible phosphorylation regulatory mechanism for IDH activity during freezing stress. Taken together, these results suggest activation and maintenance of IDH activity despite hypometabolic conditions. This initiation in activity of IDH during freezing may play a role in antioxidant defense by contributing to maintenance of the NADPH pool under stress conditions.

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Differential regulation of a gene having either canonical or non-canonical cyclic AMP response element (CRE) in its promoter is primarily accomplished by its interactions with CREB (cAMP-response element binding protein). The present study aims to delineate the mechanism of the CREB-CRE interactions at the Oncostatin-M (osm) promoter by in vitro and in silico approaches. The non-canonical CREosm consists of two half-CREs separated by a short intervening sequence of 9 base pairs. In this study, in vitro binding assays revealed that out of the two CRE half-sites, the right half-CRE was indispensable for binding of CREB, while the left sequence showed weaker binding ability and specificity. Genome-wide modeling and high throughput free energy calculations for the energy-minimized models containing CREB-CREosm revealed that there was no difference in the binding of CREB to the right half of CREosm site when compared to the entire CREosm. These results were in accordance with the in vitro studies, confirming the indispensable role of the right half-CREosm site in stable complex formation with the CREB protein. Additionally, conversion of the right half-CREosm site to a canonical CRE palindrome showed stronger CREB binding, irrespective of the presence or absence of the left CRE sequence. Thus, the present study establishes an interesting insight into the interaction of CREB with a CRE variant located at the far end of a TATA-less promoter of a cytokine-encoding gene, which in turn could be involved in the regulation of transcription under specific conditions.

A chalcone analogue, (E)-3-(phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (DMU 101), was synthesized using classic base catalysis and Claisen-Schmidt condensation, and then screened for its antidiabetic properties. The compound's effects on glucose and lipid metabolism were assayed in rats that were treated acutely and for a short time to elucidate its mechanism of action, evaluating glucose tolerance and lactate dehydrogenase activity in response to chalcone analogue administration. The chalcone's in vitro and ex vivo effects on glycogen, glucose, lipid and lipolysis were also investigated, as well as the mechanism by which it induces 45Ca2+ influx-mediated insulin secretion. The analogue (10 mg/kg) diminished glycemia, without inducing acute cell damage, increased glycogen content in the skeletal muscle and reduced serum triacylglycerol and total cholesterol, but did not alter high-density lipoprotein or low-density lipoprotein. Chalcone (10 μM) stimulated glucose uptake in the soleus muscle and did not modulate in vitro or ex vivo lipolysis. This analogue also increased insulin secretion by triggering calcium influx and blocking ATP-sensitive K+ channels and voltage-dependent calcium channels. However, it also modulated stored calcium via sarco/endoplasmic reticulum calcium ATPase (SERCA) and ryanodine receptor (RYR) activity. These findings indicate that this chalcone may induce cellular repolarization via a mechanism mediated by calcium-dependent potassium channels.

Type 2 diabetes mellitus (DM) poses a major burden for the treatment and control of tuberculosis (TB). Characterization of the underlying metabolic perturbations in DM patients with TB infection would yield insights into the pathophysiology of TB-DM, thus potentially leading to improvements in TB treatment. In this study, a multimodal metabolomics and lipidomics workflow was applied to investigate plasma metabolic profiles of patients with TB and TB-DM. Significantly different biological processes and biomarkers in TB-DM vs. TB were identified using a data-driven, knowledge-based framework. Changes in metabolic and signaling pathways related to carbohydrate and amino acid metabolism were mainly captured by amide HILIC column metabolomics analysis, while perturbations in lipid metabolism were identified by the C18 metabolomics and lipidomics analysis. Compared to TB, TB-DM exhibited elevated levels of bile acids and molecules related to carbohydrate metabolism, as well as the depletion of glutamine, retinol, lysophosphatidylcholine, and phosphatidylcholine. Moreover, arachidonic acid metabolism was determined as a potentially important factor in the interaction between TB and DM pathophysiology. In a correlation network of the significantly altered molecules, among the central nodes, chenodeoxycholic acid was robustly associated with TB and DM. Fatty acid (22:4) was a component of all significant modules. In conclusion, the integration of multimodal metabolomics and lipidomics provides a thorough picture of the metabolic changes associated with TB-DM. The results obtained from this comprehensive profiling of TB patients with DM advance the current understanding of DM comorbidity in TB infection and contribute to the development of more effective treatment.

Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with β-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.

Diabetes and ulcerative colitis are chronic diseases associated with inflammation, dysbiosis, impaired immune function and infection risk. In patients with type 1 diabetes enteropathy, gastrointestinal manifestations are seen relatively frequently. The current investigation was aimed to decipher the role of 3-aminobenzamide (3-AB) in ulcerative colitis associated Diabetes mellitus in male BALB/c mice. Ulcerative colitis associated Diabetes mellitus experimental murine model was developed by 3 cycles (each cycle consists of seven days) of Dextran Sulphate Sodium (DSS; 2.5 %w/v) with recovery time of one week in-between along with Streptozotocin (STZ; 40 mg/kg; i.p. x 5 days; consecutively) was given at the Ist recovery period. As an intervention, 3-aminobenzamide (3-AB; 5 and 10 mg/kg; intraperitoneally) was given beginning with the second DSS cycle and then continue till sacrifice. 3-aminobenzamide treatment significantly reduced the severity of colitis-associated diabetes mellitus by altering the expression of a number of molecular targets, including sirtuin 1 (SIRT 1), proliferating cell nuclear antigen (PCNA), poly[ADP-ribose] polymerase 1 (PARP-1), cysteine protease-1 (Caspase-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkBp65), NLR family pyrin domain containing 3 (NLRP3), insulin growth factor 1 (IGF-1), interleukin-1β (IL-1β), interleukin-10 (IL-10) and β-catenin. Further, 3-AB at high dose (10 mg/kg; intraperitoneally) significantly restored the epithelial tight junction integrity as evaluated by TEM analysis and restored occludin expression analysed by immunofluorescence analysis. Present study revealed that the high dose of 3-AB (10 mg/kg; intraperitoneally) showed significant and consistent protective effects against colitis associated Diabetes mellitus by modulating various molecular targets.

Protein aggregation is an underlying cause of many neurodegenerative diseases. Also, the overlapping pathological disturbances between neurodegenerative diseases and type-2 diabetes mellitus have urged the scientific community to explore potential of already available anti-diabetic medications in impeding amyloid formation too. Recent study brief out promising potential of an anti-diabetic drug Glyburide(GLY) as an inhibitor of amyloid fibrillation utilizing several biophysical techniques, computational methods and imaging tools. The mechanism of interaction was elucidated and the structural alterations in human serum albumin(HSA) as well as the microenvironment changes of its fluorophores(tryptophan, tyrosine) upon interacting with GLY were studied by spectroscopic techniques like Circular dichroism and synchronous fluorescence. Binding studies detailing about the GLY-HSA complex distance and the energy transfer efficiency was obtained by Fluorescence resonance energy transfer. For aggregation inhibition studies, the existence and size of aggregates formed in HSA and their inhibition by GLY was determined by Turbidity assay, Dynamic light scattering and Rayleigh light scattering along with dye binding assays. The ThT kinetics measurements analysis suggested that GLY deaccelerates fibrillation by decrement of apparent rate(Kapp) constant. The inhibitory effect of GLY might be attributed to native structure stabilization of HSA by obstruction into β-sheet conversion as confirmed by CD spectroscopy results. Amyloid inhibition and suppression of amyloid-induced hemolysis by GLY was further delineated by TEM and SEM analysis respectively. All these findings for the first time report the new facet of the anti-amyloidogenic potential of GLY, making it a promising candidate to treat neurodegenerative diseases too in the near future.

It is known that the activities of Na+/K+- and Ca2+-ATPases in the plasma membrane with an excess of cholesterol are compromised. Our main goal was to find out whether quercetin, resveratrol, or caffeic acid, in the nano- and low micromolar concentration ranges, can improve the ATPase activity in human erythrocyte membranes with excess cholesterol. These molecules belong to different chemical classes of polyphenols and are widely present in plant foods. Also, due to some variations in the protocol for determining the ATPase activity, we first analyzed several key parameters of the protocol to improve the accuracy of the results. The activities of Na+/K+- and Ca2+-ATPases were reduced in membranes with moderate and high cholesterol levels compared to membranes from normocholesterolemic subjects (p < 0.01). All three polyphenols affected the ATPase activity in a similar biphasic manner. Namely, the ATPase activity gradually increased with increasing polyphenol concentration up to 80-200 nM, and then gradually decreased with further increase in polyphenol concentration. Moreover, the stimulating effect of the polyphenols was highest in membranes with high cholesterol content, making ATPase activity values close/equal to those in normal cholesterol membranes. In other words, quercetin, resveratrol, and caffeic acid at nanomolar concentrations were able to improve/restore the functioning of Na+/K+- and Ca2+-ATPases in erythrocyte membranes with high cholesterol levels. This suggests a common membrane-mediated mechanism of action for these polyphenols, related to the content of membrane cholesterol.

Nuclear sialoglycans are minor components in the nucleus, and their biological significance was not well understood. Recently, Nile tilapia Neu4 sialidase (OnNeu4) was identified and reported as the first nuclear sialidase in vertebrates. Although OnNeu4 possesses the nuclear localization signal (NLS) required for nuclear localization, other fish Neu4 sialidases, such as zebrafish and Japanese medaka, also possess NLS, but their subcellular localizations are not nucleus. To understand the nuclear localization mechanism of fish Neu4, we focused on Mexican tetra Neu4 (AmNeu4), which, unlike Neu4 in other fishes, has a bipartite NLS. AmNeu4 exhibited a wide range of optimal pH and substrate specificity, and its gene expression was specifically detected in the liver, spleen, and gut in adult fish. AmNeu4, like OnNeu4, exhibited nuclear localization, which was attenuated by importin inhibitor, and deletion of the bipartite NLS completely reduced the nuclear localization. In addition, the conjugation of the bipartite NLS of AmNeu4 made GFP show nuclear localization. To understand the mechanism of nuclear localization of AmNeu4 and OnNeu4, we compared fish Neu4 amino acid sequences and focused on the less conserved region of Neu4 sialidase (LCR). LCR-deletion mutants of AmNeu4 and OnNeu4 showed significantly reduced the nuclear localization. The LCR region in AmNeu4 and OnNeu4 possessed consecutive Ser/Thr. The Neu4 mutants in which consecutive Ser/Thr in LCR were changed to Ala or deleted significantly suppressed the nuclear localization. These results suggest that the nuclear localization of Neu4 in Nile tilapia and Mexican tetra may be regulated by NLS and LCR.

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB-HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.