Zinc finger homeobox 2 (ZFHX2) is a 2572-residue-long, multidomain protein involved in the rare inherited congenital hypoalgesia. This pain insensitive phenotype is associated with a missense mutation (arginine to lysine at R1913) at the C-terminus of the second homeodomain (HDo2), comprising residues 1857-1916 of ZFHX2. In this work, we expressed and purified wild-type HDo2 and the mutant R1913K by using Ni[2+]-IMAC and a polishing gel filtration chromatography step. The isolated wild-type HDo2 was a monomeric, well-folded, stable polypeptide, with a high percentage of α-helix-like structure, as judged from the far-ultraviolet (UV) circular dichroism (CD), fluorescence and 1D-[1]H-NMR spectra. Similar conformational properties were observed for the R1913K mutant. Thermal denaturations for both species, followed by fluorescence and far-UV CD, yielded similar apparent thermal denaturation midpoints. Therefore, the development of hypoalgesia does not seem to be related to global conformational changes in the structure of the domain, as monitored by those macroscopic probes.

The rapid arrest of bleeding after injury is essential for survival, and blood coagulation is the central mechanism of hemostasis. We previously reported that a C-type lectin (HjCL) present in the skin mucus of the bullhead shark (Heterodontus japonicus) exhibits procoagulant activity; however, its underlying molecular mechanism remains unclear. In this study, we evaluated the effects of HjCL on pathway-specific clotting reactions using prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays. HjCL accelerated aPTT reagent-triggered clotting, as indicated by the shortened time to reach 50% of maximal fibrin formation (t50). In contrast, no procoagulant effect was observed in the reactions initiated by the PT reagent. We performed synthetic substrate assays for the factors Xa and thrombin to further assess the activation of common-pathway enzymes at the plasma level. HjCL enhanced thrombin generation in the aPTT assay but did not increase factor Xa generation. Together, these results suggest that HjCL does not act as an initiator of coagulation but rather promotes coagulation at a stage downstream of factor Xa formation, consistent with its role in the amplification phase of the common pathway. Our findings suggest a previously unrecognized mechanism by which a non-enzymatic protein modulates blood coagulation in cartilaginous fish, providing new insights into hemostatic regulation in early-diverging vertebrates.

Streptococcus mutans is recognized as a major etiological agent of dental caries, a highly prevalent chronic global health issue. The early identification of such cariogenic pathogens is therefore critical for implementing effective preventive strategies. To address this need, we established an integrated detection platform coupling recombinase-aided amplification (RAA) with CRISPR-Cas13a for S. mutans detection and evaluated its performance against conventional polymerase chain reaction (PCR). The proposed RAA-CRISPR-Cas13a assay exhibited superior sensitivity and faster turnaround time compared to PCR. A distinctive feature of the assay design was the inclusion of a T7 promoter sequence within the forward primer, thereby facilitating direct in vitro transcription without requiring an additional oligonucleotide. The assay demonstrated a broad quantitative detection range from 10[1] to 10[9] CFU/mL, with a limit of detection (LOD) reaching 3.2 CFU/mL. A strong linear relationship (R[2] > 0.98) was established between the fluorescence response and the logarithmic bacterial concentration, underscoring the assay's accuracy and robustness. This sensitive and rapid detection system shows considerable promise for screening high-risk populations, allowing not only early detection but also potential evaluation of caries severity and progression. Its simplicity and minimal instrumentation requirements make it particularly suitable for low-resource healthcare environments.

Parasitic nematodes inflict billion-dollar losses globally each year as they infect humans, livestock and plants. Exploiting fundamental biochemical differences between these parasites and their hosts could accelerate the discovery of improved control measures. One key area of biochemical difference between nematodes and their hosts is sterol biosynthesis as nematodes, unlike vertebrates, nematodes are unable to synthesise sterols de novo and therefore depend on exogenous sterols obtained from their diet or environment. However, they can extensively modify these sterols through specialised metabolic pathways, producing metabolites including C4-methylated sterols and dafachronic acids that have important roles in nematode development and physiology. This mini review focuses on sterol biochemistry and metabolism of nematodes, in particular, sterol metabolism in the model worm Caenorhabditis elegans.

This review on autism provides a summary of the multiple genetic, epigenetic and environmental changes setting the stage for disturbed brain development due to aberrant synaptogenesis and corticogenesis during the prenatal and post-natal periods. The emphasis is placed on nonmendelian transmission driven by epigenetics. Folate deficiency appears to play a critical role in influencing many of the structural changes contributing to developmental deficits. Impaired passage of folate across the placental and the blood-brain barriers to the fetal and postnatal brain because of folate receptor autoantibodies (FRAb) emerges as the major cause. Cerebral folate deficiency with or without autism has been associated with FOLR1 gene defects, a rare disorder. On the other hand, FRAb in association with low CSF folate is present in most children diagnosed with autism. These autoantibodies are present in more than 1/3 of asymptomatic parents of children with autism or are acquired by the child during the first two years of life. Early detection of FRAb amongst autistic children is necessary to enable high dose folinic acid treatment. The use of folinic acid before conception and during pregnancy among mothers or both parents with FRAb, represents a promising strategy to reduce the risk of autism in future offspring. Interventational treatments in parents prior to pregnancy and optimal therapeutic approaches in infants are needed to prevent disordered development of the pre- or post-natal brain.

Neuroinflammation is a central pathogenic mechanism in numerous neurological disorders, with microglia playing a pivotal role in coordinating immune responses through tightly regulated redox, metabolic, and inflammatory signaling pathways. While transient microglial activation supports tissue homeostasis and repair, persistent activation promotes excessive production of reactive oxygen and nitrogen species (ROS/RNS), pro-inflammatory mediators, and lipid-derived inflammatory signals, driving chronic neuroinflammation and neurodegeneration. Accordingly, modulation of microglial activation has emerged as an attractive therapeutic strategy. Plant-derived extracts and phytochemicals represent a remarkable source of bioactive molecules such as quercetin, which are able to concomitantly modulate multiple pathways involved in microglial activation. Over the past two decades, numerous studies have investigated their effects using immortalized murine BV-2 cells and, to a lesser extent, primary microglia. Despite differences between these experimental models, current evidence reveals a striking convergence of molecular mechanisms. Plant-derived metabolites consistently attenuate microglial activation through inhibition of the NF-κB and MAPK signaling pathways, activation of the Nrf2/HO-1 antioxidant axis, suppression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokine production, while limiting oxidative stress and lipid peroxidation. Emerging evidence further indicates that these compounds regulate microglial phenotypic plasticity, mitochondrial function, immunometabolic reprogramming, and, potentially, peroxisomal metabolism and mitochondria-peroxisome crosstalk, thereby extending their actions well beyond conventional antioxidant activity. This review critically examines the molecular mechanisms by which plant-derived extracts and phytochemicals regulate microglial function, with particular emphasis on the interconnected networks linking redox homeostasis, inflammatory signaling, lipid metabolism, and organelle communication.

Fibrinolytic enzymes of microbial origin are gaining attention as safer alternatives to conventional thrombolytic drugs. In this study, fibrinolytic enzymes were isolated and characterized from Bacillus subtilis and Bacillus thuringiensis derived from soil samples. Process optimization revealed maximum enzyme production at 37°C and pH 7.0 after 24 h of incubation, with inoculum sizes of 1.5% (B. subtilis) and 1% (B. thuringiensis) in media supplemented with glucose/sucrose and yeast extract. Partial purification yielded specific activities of 191.15 U/mg (B. subtilis) and 218.24 U/mg (B. thuringiensis) with recovery yields of 14.39% and 13.89%, respectively. Both enzymes exhibited stability over broad ranges of pH (5.0-9.0) and temperature (10-60°C). Enzyme activity was enhanced by Mg[2+] and Ca[2+] but completely inhibited by EDTA and PMSF, suggestive of serine metalloproteases classification. Amidolytic assays demonstrated high activity with a chymotrypsin substrate, while hemolytic activity remained below 5%, indicating the possibility of initial therapeutic safety. SDS-PAGE revealed molecular masses of 35 kDa (B. subtilis) and 33 kDa (B. thuringiensis). Kinetic analysis showed Km values of 3.488 mg/mL and 3.125 mg/mL with Vmax values of 232.56 U/mL and 178.57 U/mL, respectively. These findings suggest that fibrinolytic enzymes from both strains exhibit promising activity and stability profiles and provide a basis for future studies to determine their potential role in thrombolytic drug development..

Among nutrient-sensitive regulatory mechanisms, protein glycosylation is a key post-translational modification that links metabolism to protein function. Amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer's disease (AD). Because excessive accumulation of Aβ is believed to drive AD progression, approaches that reduce Aβ production and secretion remain of considerable therapeutic interest. Accumulating evidence suggests that protein glycosylation influences the processing and trafficking of amyloid-β precursor protein (APP) and other proteins involved in Aβ metabolism. However, while N-linked protein glycosylation has been shown to affect Aβ production, the specific contribution of protein terminal glycan sialylation remains incompletely understood. In this study, we investigated the effects of inhibiting glycan sialylation on Aβ production using CHO cells stably expressing human APP. Treatment with the sialyltransferase inhibitor P-3Fax-Neu5Ac (P-3Fax) significantly reduced extracellular Aβ levels, while intracellular Aβ levels were increased. Importantly, P-3Fax treatment did not alter β-secretase- or γ-secretase-mediated cleavage of their substrates, indicating that changes in secretase activity do not account for the reduction in extracellular Aβ. In addition, P-3Fax treatment markedly reduced plasma membrane levels of lysosome-associated membrane protein 1 (LAMP1), consistent with impaired lysosomal exocytosis. Taken together, these results indicate that inhibition of sialylation by P-3Fax reduces extracellular Aβ levels independently of secretase activity, potentially through impaired lysosomal exocytotic pathways. Our findings identify glycan sialylation as a key regulatory factor in Aβ trafficking and provide new insight into the cellular mechanisms controlling Aβ homeostasis.

Ether glycerophospholipids are abundant and functionally versatile membrane lipids whose defining sn-1 ether (plasmanyl), or vinyl ether (plasmenyl, plasmalogen) linkage, and polyunsaturated sn-2 chains provide membranes with unique biophysical and signaling properties. Biogenesis is partitioned between two organelles: peroxisomes establish the sn-1 ether linkage and deliver 1-O-alkyl intermediates to the endoplasmic reticulum, where headgroup addition and remodeling occur. Introduction of the vinyl ether double bond by plasmanylethanolamine desaturase then yields plasmalogens. Emerging evidence indicates that ether lipids are unevenly distributed across cellular membranes, reflecting both local metabolic control and selective trafficking. Biosynthetic intermediates are delivered from peroxisomes to the endoplasmic reticulum via ACBD4/5-VAPA/B membrane contact sites, which also couple ether lipid-flux to cholesterol homeostasis. At the plasma membrane, ATP8B2 flippase enforces inner leaflet enrichment of plasmalogens, linking their molecular shape to membrane curvature, raft organization, and signaling. Beyond these core sites, additional pools of ether lipids are detected in the Golgi, mitochondria, endolysosomal system, lipid droplets, and the nucleus, where they are proposed to support membrane organization, and trafficking, yet their organelle-specific functions and transport mechanisms remain poorly defined. In this review we summarize the current knowledge about cellular and subcellular distribution of ether lipids, their biosynthesis, remodeling, degradation, and intracellular trafficking. We also highlight recent methodological advances enabling organelle-resolved lipidomics and live-cell tracking of ether lipids and outline open questions regarding contact-site identity and regulation, transport specificity between plasmanyl and plasmenyl species, and pathophysiological mislocalization mechanisms.

Among the various proteins found in the venom of snakes belonging to the genus Bothrops, which is responsible for the highest number of snakebite accidents in Brazil, Phospholipase A2 (PLA2) is one of the most important components. The present study demonstrated the isolation of a fraction containing two Asp49 PLA2 isoforms from the endemic southeastern Brazilian snake Bothrops fonsecai, named BfPLA2. The isolation was achieved through two chromatographic techniques: RP-HPLC using a Teknokroma Europa Protein 300 C18 column, followed by anion-exchange chromatography using a HiTrap Q FF column. SDS-PAGE and two-dimensional electrophoresis revealed proteins with an approximate molecular mass of 15 kDa and an isoelectric point close to 5, indicating acidic PLA2 isoforms. Mass spectrometry analysis and bioinformatic analysis identified two isoforms with high similarity to PLA2s previously described in literature, corroborating the protein family of the isolated toxins. BfPLA2 showed maximum activity at 60 °C and pH 8.5 (Mean: 223.89 U/min/mg). Cytotoxic activity against the HeLa cell line was demonstrated (IC50: 26.21 μg/mL), as well as anticoagulant activity, prolonging coagulation time by 74.1% at the highest dose, and hemolytic activity (IC50: 8.26 μg/mL). The characterization of proteins isolated from snake venoms may contribute to a deeper understanding of snakebite envenomation, especially regarding Bothrops fonsecai, whose venom remains poorly characterized.

Phosphine-releasing metal phosphides are used to protect stored grains. Phosphine gas can be fatal if inhaled. However, the easy availability of metal phosphides (like Aluminium phosphide) causes increased misuse to commit suicide. Phosphine is a mitochondrial poison and affects the body's major organs. Phosphine poisoning has no specific diagnostic biomarker or remedy/antidote. It is reported that phosphine inhibits erythrocyte cholinesterase activity. However, contradictory views regarding this issue exist, and no consensus has been reached yet. Further, it is completely unknown that even if phosphine inhibits erythrocyte cholinesterase activity, such inhibition is reactivated by standard oxime reactivators akin to organophosphorus pesticide-induced enzyme inhibition. Keeping in mind that there is no antidote to phosphine exposure, it is important to solve the reactivation issue in the context of phosphine-induced enzyme inhibition. The in-silico (molecular docking and molecular dynamics simulation studies) and in-vitro (electrophoretic and spectroscopic) studies were carried out to understand the effect of phosphine exposure on erythrocyte cholinesterase activity. Besides the oximes used to understand the reactivation of the enzyme activity. From the results, it was discovered that phosphine inhibits human erythrocyte cholinesterase, akin to neostigmine and dichlorvos. Moreover, such inhibition is reactivated by oximes. It is concluded that erythrocyte cholinesterase activity holds significant promise as a biomarker for phosphine inhalation toxicity, and oximes may serve as a potential antidote.

The cell membrane plays a critical role in maintaining cellular integrity and regulating interactions between the cell and its environment. The understanding of many biological processes relies on obtaining molecular level information on the structure and dynamic behaviour of membranes, interactions between molecules within the membrane, as well as interactions between membrane and external compounds. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy has emerged as a powerful analytical technique for investigating biological membranes due to its surface sensitivity and minimal sample preparation requirements. The technique enables the characterization of lipid bilayers, protein secondary structures, and membrane interactions with external molecules such as drugs or antimicrobial peptides. This mini-review summarizes the principles of ATR spectroscopy and highlights its applications in the study of lipid bilayers, including lipid organization, protein-lipid interactions, and membrane structural changes. The advantages, limitations, and future perspectives of ATR-based approaches in membrane research are also discussed.

Marine Aspergillus terreus is explored as an important chitinase-producing fungal strain for N-Acetyl-D-Glucosamine (GlcNAc) production from chitin substrates. Here, a purified extracellular 45 kDa chitinase of marine Aspergillus terreus (accession number JQ248076) was characterized in terms of substrate specificity. Conventionally, endochitinase cleaves chitin randomly to produce GlcNAc and its various multimers. whereas, exochitinases cleave the chitin from its reducing end and produce either GlcNAc or chitobiose (GlcNAc dimer). In the present chitinase characterization, the HPLC followed by HRMS analyses revealed differential product formation from the chitin substrates of varied chain length. With swollen chitin , the enzyme produced GlcNAc as a predominant product; whereas with chitohexaose substrate, a mixture of GlcNAc and its oligomers was obtained. Although mass spectrometry-based proteomics analysis identified the isolated chitinase as an endochitinase 1 precursor (Accession XP_001217186). However, the enzyme kinetic study exhibited higher catalytic efficiency for exochitinase-specific dimeric chromogenic substrate in comparison to endochitinase-specific tetrameric fluorogenic substrate, which indicated predominantly exochitinase behaviour of the enzyme. Further, the in silico study predicted a differential cleavage pattern of the enzyme, which could be due to different mode of substrate binding and processive mechanism through the tunnel-shaped binding cleft of the enzyme. The substrate specificsubstrate specific mode of catalytic activity of present chitinase was further confirmed by a molecular docking study with different lengths of substrates. With unique substrate specific substrate specific mode of action, the chitinase of marine Aspergillus terreus offers a great promise for the production of GlcNAc.

Obesity remains a global pandemic and a major driver of metabolic diseases, with current pharmacotherapies often limited by modest efficacy, systemic side effects, high costs, and weight regain after cessation. Targeting dietary lipid digestion via the inhibition of gastrointestinal lipases offers a mechanistically rational and peripherally acting therapeutic strategy. This review provides an updated, quantitative overview of human gastrointestinal lipolysis, emphasizing the sequential and complementary roles of human gastric lipase and human pancreatic lipase in dietary triglyceride absorption. We critically analyze the clinical proof-of-concept provided by Orlistat, the only approved digestive lipase inhibitor, whose utility is constrained by limited efficacy, significant gastrointestinal intolerance, and rare but severe hepatotoxicity. While next-generation systemic therapies like GLP-1 receptor agonists show superior weight loss, their chronic administration and high cost underscore the need for safer, localized alternatives. We systematically review recent strategies for developing superior digestive lipase inhibitors, including structure-based drug design, in silico screening, and bioguided fractionation of natural products (e.g., flavonoid derivatives). Emphasis is placed on the critical design principles required for next-generation inhibitors, including strict luminal confinement, high selectivity for digestive lipases, and controlled, reversible inhibition to improve tolerability. By bridging biochemical insights with pharmacological challenges, this review outlines a roadmap for the development of safer, more effective lipid-targeted therapies to combat obesity.

Sulfoquinovosyldiacylglycerol (SQDG) is an anionic sulfolipid of plastid membranes, classically associated with chloroplasts and thylakoids. Its detection by lipidomics in chloroplast-poor tissues raises a central problem: how can a reported SQDG signal be validated, and what evidence is required to determine its plastid context and biological meaning? In this critical narrative review, we reassess evidence beyond chloroplast-dominated tissues and evaluate both the strength of evidence for SQDG occurrence or function and the steps required to test lipidomics-derived hypotheses. The literature supports context-dependent rather than universal interpretations. Direct organelle-level lipid evidence from isolated non-green plastids remains limited; etioplast genetics supports an SQDG-related role in non-photosynthetic plastid membrane development; phosphate-deprived roots support phosphorus-economy remodeling; and sulfur-starvation studies establish regulated SQDG turnover and sulfur remobilization. By contrast, chromoplast and woody-tissue interpretations remain unresolved. These findings show that SQDG abundance alone is hypothesis-generating rather than diagnostic of function. We therefore propose a decision-tree framework that moves from chemical confirmation of the lipid signal to spatial localization, SQD1/SQD2-associated biosynthetic capacity, nutrient context, tissue-specific perturbation, and phenotype testing. This framework provides criteria for moving from lipidomic detection to supported biological interpretation in chloroplast-poor tissues.