The cytokine tumor necrosis factor (TNF), a major regulator of inflammatory responses, exists in both a membrane bound form and a soluble form. We used  the nonselective TNF inhibitor etanercept, and the selective inhibitor XPro1595 and compared supraspinatus muscle cytokine levels, histology and proteomic signatures in mice after supraspinatus tendon tear. The aim was to investigate the effect of anti-TNF treatment in the early inflammatory response in the muscle after tendon tear. In addition, the effect on body composition and bone mineral content were compared in naïve mice after two of treatment with either etanercept or XPro1595 using DEXA and micro-CT. Inhibition of TNF did not significantly affect DEXA indexes of body composition nor bone microarchitecture, apart from increased structure model index and decreased bone surface density at 14 days, and bone surface to volume ratio at two months.  Supraspinatus tendon tear caused extensive inflammatory changes in the supraspinatus muscle and initiated a regenerative response. However, TNF-inhibition did not significantly impact these processes recorded as  density in the lesioned supraspinatus muscle of macrophages and myogenin positive nuclei. Though  both inhibitors had an effect on mitochondrial proteins, particularly etanercept tended to modulate mitochondrial function, and eternacept also influezed  NF-κB signaling. Modulation of mitochondrial proteome, and the  influence on  NF-κB signaling seen after etanercept treatment could correspond with its known effect on apoptosis.

The two murine double minute (MDM) family members, MDM2 and MDMX, are a well-established negative regulator of p53 activity. Under DNA damage conditions, MDM2 and MDMX are phosphorylated near their RING domains (serine 395 at MDM2 and serine 403 at MDMX) and switch to act as p53 positive regulators. MDMX binds to TP53 mRNA and acts as a chaperone for RNA structure, enabling MDM2 to bind. This interaction enhances TP53 mRNA translation, leading to increased p53 protein production. While the biological significance of this interaction has been described, the specific features of the MDMX-RNA interaction remain poorly understood. We used various MDMX protein constructs to characterize binding to TP53 mRNA and identified that the interaction mediated by the RING domain is modulated by the presence of other domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and binding assays in high salt conditions and various pH demonstrate that the whole protein participates in RNA interaction, with the C-terminal domain likely providing the contact with RNA by electrostatic forces. We show that protein structural changes induced by the chelating agent EDTA or the reducing agent TCEP enhance RNA binding by promoting partial structural destabilization of the protein. Our findings suggest that the MDMX/TP53 mRNA interaction is complex, with the RING domain binding to RNA and being supported by the entire protein, which acts as a scaffold for the RNA interaction. These results contribute to a better understanding of MDMX's role in TP53 mRNA binding and provide valuable insights for future investigation of the MDM2-MDMX-TP53 mRNA complex, which is crucial for p53 stabilization and activation under DNA-damaging conditions.

The nematode Caenorhabditis elegans biosynthesizes the ascarosides, a large, modular family of pheromones that are used in chemical communication. A number of carboxylesterase-like (CEST) enzymes are responsible for decorating the glycolipid core of the ascarosides with a variety of modifications. However, these enzymes, which are homologous to carboxylesterases and acetylcholinesterases, have not been characterized biochemically, and thus the mechanism whereby they attach different modifications to the ascarosides is unknown. Here, we report the expression, purification and biochemical characterization of soluble CEST enzymes for the first time. In this study, we focused on CEST-9.2, which is responsible for making (E)-2-methylbut-2-enoyl (MB)-modified ascarosides. We identified candidate substrates for the enzyme, and we successfully expressed a truncated version of CEST-9.2 in several expression systems, including Escherichia coli, Pichia pastoris and Spodoptera frugiperda Sf9 cells. The purified CEST-9.2 from each of these systems was tested against candidate substrates, including ascarosides and either MB-carnitine, MB-choline, or MB-coenzyme A (CoA). However, no enzymatic activity was detected using these substrates, suggesting that either the transmembrane domain is necessary for activity or that the correct substrates have not yet been identified.

Human ecdysoneless (ECD), the human ortholog of Ecd protein of Drosophila melanogaster, is a highly conserved protein. This protein comprises a well-folded N-terminal domain and a disordered C-terminal domain; these domains interact with multiple proteins and are involved in diverse biological processes, including cell cycle regulation, transactivation, pre-mRNA splicing, and glucose metabolism. ECD is highly expressed in various cancers, and its elevated expression is associated with poor prognosis in several types of human cancers. Therefore, it can serve as a potential biomarker and therapeutic target for treating tumors. This review focuses on the currently available knowledge of the physiological and pathological functions of ECD; moreover, the directions of prospective research in the relevant field have been discussed.

FGR proto-oncogene (Fgr), a member of the Src family kinases, has garnered attention for its potential involvement in apoptotic signaling, yet its role in cardiovascular diseases, particularly acute myocardial infarction (AMI), remains unexplored. This study sought to investigate whether elevated left ventricular Fgr expression alleviates myocardial injury in the infarcted area and whether this protective mechanism is mediated by modulating phosphoinositide 3-kinase (PI3K)/Akt phosphorylation. The transcriptome-wide association study was initially utilized to screen for susceptibility genes in the left ventricle, with findings validated using bulk-RNA sequencing data from a rat model of left anterior descending coronary artery (LAD) ligation; subsequently, human spatial transcriptomics combined with single-nucleus RNA sequencing data confirmed differential expression of Fgr and PI3K/Akt in the infarcted region. Fgr knockdown via siRNA in H9C2 cells and pharmacological inhibition with TL02-59 in rats were conducted to assess cellular survival and cardiac function, respectively. Fgr emerged as a common candidate gene identified through multi-omics data analysis, with its up-regulation confirmed both in vivo and in vitro. Fgr silencing in an in vitro oxygenglucose deprivation model significantly reduced cell survival and suppressed PI3K/Akt phosphorylation, whereas TL02-59 administration in rats subjected to LAD ligation impaired post-infarction cardiac function while concurrently inhibiting PI3K/Akt phosphorylation levels. This study demonstrates that Fgr is markedly up-regulated in AMI and exerts cardioprotective effects, possibly through modulation of PI3K/Akt signaling phosphorylation, thereby underscoring its potential as a therapeutic target.

Polysaccharides have gained considerable attention recently because of their anti-tumor and immunoregulatory properties. Its activity depends on the type of fungus that produces it, the extraction method, and the molecular weight.

Apolipoprotein B mRNA editing catalytic subunit 3B (A3B), a nuclear enzyme that catalyzes cytidine-to-uridine (C-to-U) editing in single-stranded DNA (ssDNA), contributes to genetic diversity in many cancers. A3B is induced or activated by DNA damage owing to a variety of factors; however, the mechanisms by which A3B accesses ssDNA within the genome remain unclear. In this study, we showed that in unstimulated cells, A3B is retained in the nucleoplasm in an RNA-dependent manner. Upon DNA damage induced by camptothecin or actinomycin D (Act D), both targeting topoisomerase I, or by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), an alkylating agent that generates apurinic/apyrimidinic sites, A3B accumulates at the nucleolar rim and interior. Using confocal microscopy, we assessed the colocalization of A3B with drug-induced R-loops. A3B accumulation was abolished by RNase H treatment, implicating R-loops in its localization. However, the S9.6 antibody, commonly used to detect DNA/RNA hybrids, did not identify R-loop-specific signals in the nucleolus, leaving the direct involvement of R-loops in A3B accumulation unresolved. Conversely, immunoprecipitation-mass spectrometry with data-independent acquisition (IP-MS DIA) revealed increased interactions between A3B and RNA helicases such as DDX17 and DDX21, which are known R-loop-binding proteins, following MNNG or Act D treatment. Our results demonstrate that A3B-induced secondary DNA damage occurs in the nucleolus after DNA damage, providing new insights into the acquisition of cancer diversity involving A3B and the DNA damage response in the nucleolus.

Proteome, the molecular product of regulatory diktat of the cellular machinery, predicts the behaviour and progression of cancers. Designing effective molecular therapies based on proteins with comprehensive patient stratification remains the mainstay of every translational research. Research on the proteome involves a) identification of biomarkers that, with utmost sensitivity and specificity, reveal significant insights into the disease state and b) understanding the mechanistic underpinnings and rewiring of cellular signaling pathways that drive a particular cancerous pathology. In this review, we give a comprehensive description of the evolution of mass spectrometer-based methods, including labeling strategies available to study the proteome and post-translational modifications in response to various perturbations. We summarize their utility in understanding complex processes of cancers, advance research on cancer therapy by decoding novel biomarkers, identify therapy resistance drivers, and enhance spatial attributes of tumor microenvironment by single-cell proteomics. Finally, some of the challenges in the currently used methods have been discussed.

Diaminopimelate decarboxylase (DAPDC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the decarboxylation of diaminopimelate (DAP) to yield L-lysine, a key step in lysine biosynthesis. This present study presents a preliminary characterization of DAPDC encoded by the cce1351 gene in Cyanothece sp. ATCC 51142 (CsDAPDC), focusing on its biochemical properties and model structure characteristics. The enzyme exhibited a peak activity at 30°C and pH 8.0, and the catalytic constant (kcat) and substrate binding affinity Michaelis constant (KM) were determined as 1.68 s-1 and 1.20 mM at the above-mentioned condition, respectively. Homology modeling and molecular docking analysis revealed that Gly286, Gly330, Tyr428, and Asp118 interacted with the PLP cofactor, and Ser249, Tyr372, and Tyr428 interacted with the DAP substrate. Additionally, Cys399, Glu400, and Tyr436 from the other monomer were also involved in binding DAP and PLP. Site-directed mutagenesis confirmed the functional roles of these key residues in catalysis. This work provides valuable insights into the catalytic mechanism of CsDAPDC and highlights the enzyme's potential for applications in metabolic engineering of cyanobacteria for enhanced lysine production.

Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries. Early detection remains challenging, with no established plasma-based biomarkers for clinical use. This study aimed to evaluate plasma adipokines and their receptor expression as diagnostic biomarkers for EC. Plasma levels of leptin, soluble leptin receptor, visfatin and asprosin were quantified in EC and control patients using ELISA. The free leptin index (FLI) was calculated as a ratio of leptin to soluble leptin receptor. Gene expression of corresponding receptors, including leptin receptor (Ob-R), insulin receptor (INSR), glucagon-like peptide-1 receptor [GLP-1 receptor (GLP-1R)], and asprosin-associated receptors, toll-like receptor 4 (TLR4), protein tyrosine phosphatase receptor type D (PTPRD), and olfactory receptor family 4 subfamily M member 1, was assessed by RT-qPCR from total blood. Plasma leptin levels were significantly elevated in EC patients, with the FLI over four times higher than controls (P=0.008). Soluble leptin receptor levels trended lower in EC, though non-significantly. Visfatin and asprosin plasma levels showed non-significant elevations. Gene expression analyses revealed significantly increased levels of GLP-1R, TLR4 and PTPRD in EC patients, suggestive of a diagnostic potential. Notably, plasma biomarker levels were not independently correlated with body mass index (BMI). Elevated FLI and up-regulation of adipokine receptor expression highlight the potential of combining plasma-based and molecular biomarkers for EC diagnosis. However, the lack of independence from BMI and conflicting literature underscores the need for larger, standardised studies to validate these findings and determine clinical applicability.

Ever since its discovery more than 70 years ago, the enzyme polynucleotide phosphorylase (PNPase) has been the subject of intensive research that has highlighted its key functional roles. The enzyme was first described in 1955 for its ability to synthesise RNA from nucleoside diphosphates. This discovery led to a Nobel Prize in Physiology or Medicine in 1959 for using PNPase to synthesise artificial RNA. However, it soon became evident that the primary function of this enzyme, conserved across diverse species, is 3'-5' RNA phosphorolysis rather than polymerisation. Remarkably, over 60 years later, it was discovered that PNPase has an even broader range of functions as it was shown to act as a conditional RNA chaperone in bacteria. In humans, PNPase (hPNPase) is located in mitochondria, where it plays a role in mitochondrial RNA (mtRNA) metabolism, thereby regulating mitochondrial function and the overall cell fitness. In this review, we present the current scope of knowledge of hPNPase, including its structure, subcellular localisation, metabolic activity, roles in mtRNA transport, processing and degradation, and its involvement in apoptosis.

Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.

Brain-expressed voltage-gated sodium (Nav) and potassium (Kv) channels are essential for maintaining the balance of neuronal excitability, each having opposing effects on membrane potential and neuronal firing. Genetic alterations in these channels can disrupt this balance, leading to epilepsy and/or developmental impairments through gain-of-function (GoF) or loss-of-function (LoF) mechanisms. This review catalogs 48 transgenic mouse models involving sodium channels (SCN1A, SCN2A, SCN3A, SCN8A) and potassium channels (KCNQ2, KCNQ3, KCNT1, KCNA1, KCNB1, KCND2), detailing the effects of genetic alterations in terms of channel function, affected cell types, and phenotypic manifestations. Mechanistic insights from these models reveal that initial channel dysfunction triggers cascading pathological processes including glutamate excitotoxicity, oxidative stress, gliosis, neuroinflammation, and blood-brain barrier disruption. Therapeutic approaches include antisense oligonucleotides to enhance functional allele expression or reduce pathogenic channel expression, viral-mediated gene therapy, gene editing, and small molecule modulators that target persistent sodium currents or that stabilize channel inactivation. The timing of intervention appears to be critical, with early treatment showing greater efficacy in preventing pathological cascades. Strain-specific background effects and compensatory ion channel expression affect phenotypic severity and treatment response, complicating translation of model results. Importantly, transgenic models offer opportunities to better understand mechanisms underlying comorbidities commonly suffered by patients, including behavioral disorders, motor impairments, and sleep disturbances. The integration of these findings suggests that effective treatment strategies may require combinations of channel-directed therapies and interventions targeting downstream pathological processes, particularly for established disease. This comprehensive examination of channelopathy models provides a framework for developing transformative therapeutics for genetic epilepsies.

Diabetes mellitus is a complex metabolic disorder associated with severe complications affecting various organs, including the kidneys, nerves, heart, and blood vessels. Managing these complications remains a significant clinical challenge, necessitating the exploration of novel therapeutic approaches. This review focuses on polydatin, a naturally occurring glycoside from Polygonum cuspidatum, highlighting its potential as a multitargeted therapeutic agent against diabetic complications. Evidence indicates that polydatin effectively improves insulin sensitivity, lowers blood glucose levels, and exhibits antioxidant properties. In diabetic nephropathy, polydatin has been shown to reduce oxidative stress, inflammation, and podocyte apoptosis, thereby preserving renal function. Furthermore, it enhances mitochondrial function and Sirt1 expression in diabetic neuropathy, promoting nerve regeneration and alleviating pain. In cardiac studies, polydatin protects against diabetic cardiomyopathy by enhancing autophagy and reducing oxidative stress, ultimately improving cardiac function. Additionally, polydatin restores endothelial function in vascular complications associated with diabetes. Polydatin presents a promising natural therapy with the potential to mitigate multiple complications of diabetes through its antioxidant, anti-inflammatory, and cytoprotective effects. Although findings from animal models and in vitro studies are promising, further clinical research is essential to validate its efficacy and safety in human subjects. By integrating polydatin into diabetes management strategies, there is potential for improved health outcomes and quality of life for individuals affected by this chronic condition.