Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating and currently untreatable neurodegenerative diseases, whose genetic and molecular etiologies remain largely unclear. The histopathological hallmark of both diseases is the cytoplasmic deposition of TDP-43 in neurons, which is attributed to both intrinsic (e.g., mutations, aberrant cleavage) and extrinsic factors (e.g., prolonged oxidative stress, impaired clearance pathways). Mutations and certain PTMs (e.g., cysteine oxidation) destabilize RNA binding, promoting monomer misfolding and increasing its half-life. Disruptions to core ubiquitin-proteasome system (UPS) subunits impede efficient processing, contributing to the clearance failure of misfolded TDP-43 monomers. The accumulation of monomers drives phase separation within stress granules, creating nucleation hotspots that eventually bypass the thermodynamic barrier, resulting in exponential growth. This rapid growth then culminates in the failure of the autophagy-lysosome pathway (ALP) to contain the aggregation, resulting in a self-sustaining feed-forward loop. Here, we synthesize these factors into a unified kinetic cascade model. Therapeutic strategies must therefore move beyond simple clearance and focus on targeting these kinetic inflection points (e.g., oligomer seeding, PTM modulation).

Abstract: Despite decades of investigation, Aldose Reductase (AR; AKR1B1) -an enzyme that plays a key role in the metabolism of glucose and other carbonyl compounds and whose hy-peractivity contributes to oxidative stress and vascular dysfunction- inhibitors have failed to translate into clinical application for Diabetic Retinopathy (DR). We argue that these failures might arise from non-selective inhibition, which does not consider AR’s dual roles in pathology but also in retinal health, as AR is also an important detoxifying enzyme for aldehydes produced during oxidative stress, and discuss the missing structural infor-mation, despite the over one hundred crystal structures of AR in complex with inhibitors. Our review bridges this gap by proposing how recent advances in structural biology, namely, fragment-based drug discovery and MicroED, provide novel ways of selectively modulating AR functions, offering advantages in the detection of weak, allosteric, or conformation-dependent binding events. Despite past challenges, we suggest that therapeutic targeting or finding new-generation inhibitors for AR will become more effective once we have a clearer understanding of AR’s requirements for selective inhi-bition of its pathological and physiological functions. By integrating fragment screening and structural biology, we outline a strategy to reinvigorate AR modulation as a viable retina-specific approach for managing DR first, although potentially relevant across multiple diabetic microvascular complications later.

Abstract: Antimicrobial resistance (AMR) poses a global health threat, with urban wastewater systems serving as key reservoirs for resistance dissemination. This study aimed to investigate the relationships among urban environments, bacterial communities, and AMR patterns, and evaluate the specific municipal-scale drivers of resistance gene distribution. Shotgun metagenomic analysis was conducted on 45 wastewater samples collected from 15 municipalities across Latvia to determine the composition of the resistome and its correlation with local factors. The analysis identified 417 distinct antibiotic resistance genes (ARGs) belonging to 108 families, with geographic location serving as the primary driver of ARG distribution, which explained 65.87% of community variation (p = 0.001). Local industrial factors demonstrated significant effects, with food industry wastewater significantly influencing both bacterial taxonomy and ARG profiles (p < 0.05). While the presence of a regional hospital did not shape the overall municipal resistome, hospital-associated wastewater showed 19 overlapping ARGs, including clinically critical carbapenemases. Municipal wastewater systems function as geographically structured reservoirs of AMR that are shaped by localized industrial and healthcare outputs. These findings support wastewater-based AMR surveillance as a valuable tool for tracking specific resistance sources.

Abstract: In this work, we analyzed the ability to incorporate 8-oxo-dATP by several human DNA polymerases: replicative Pol ε (exo-), BER enzymes Pol β and Pol λ, and trans-lesion Pol η, Pol ι, and Pol κ. We demonstrated that human DNA polymerases differ in their abilities to discriminate against 8-oxo-dATP. Among tested DNA polymerases, Pol λ demonstrated the worst ability to discriminate against 8-oxo-dATP opposite template T on both singles-stranded DNA and double-stranded DNA substrates with a 1 nt gap. In contrast, Pol β was quite accurate on singe-stranded DNA substrate but incorporated 8-oxo-dATP opposite template T in a 1 nt gap. Unexpectedly, the catalytic subunit of high-fidelity Pol ε (exo-) incorporated 8-oxodATP opposite templates T and G with weak but higher efficiency compare to error-prone polymerases of Family Y. While structures of human polymerases with incoming 8oxo-dATP are not available, we speculate possible mechanism of 8-oxo-dATP discriminations.

Abstract: L-Glutamate (L-Glu) is the major excitatory neurotransmitter in the central nervous system and plays a key role in neuronal communication, energy metabolism, and cellular development. However, excessive glutamatergic transmission can induce excitotoxicity, leading to neuronal damage and death. Beyond its physiological role, L-Glu, commonly used in the food industry as monosodium glutamate (MSG), has raised safety concerns due to its potential adverse effects, highlighting the importance of L-Glu detection in biological and food samples. In this work, we investigate the binding interactions between the glutamate-binding protein (GluB) from Corynebacterium glutamicum and L-Glu under different pH conditions using fluorescence correlation spectroscopy (FCS). GluB was labeled with CF488 and CF647 dyes, and fluorescence fluctuations were analyzed in the absence and in the presence of L-Glu. Steady-state fluorescence measurements were conducted on the unlabeled GluB, supporting FCS. They revealed pH-dependent structural changes of GluB, with conformational rearrangements at acidic pH and partial denaturation at alkaline conditions. At pH 8.0, GluB displayed a stable conformation and a measurable response to L-Glu binding. Moreover, experiments performed on the near-infrared labeled GluB-CF647 suggested a potential applicability of GluB for living cell studies.

Abstract:

From a biochemical perspective, the recently introduced collective term luciferase-like monooxygenase family (LLM family) is notable for grouping together bacterial enzymes with fundamentally different functional characteristics. Thus not only does the family include non-bioluminescent and well as bioluminescent enzymes, but additionally both anoxybiontic and oxybiontic enzymes. By reviewing both the relatively short history of the LLM family itself, and the more protracted development of our present understanding of a number of the biochemically disparate composite enzyme groups, alternative representational descriptors can be identified that better serve both to succinctly delineate and functionally characterise the discrete groups currently corralled into the LLM family.

Abstract: Background: Endometriosis is a systemic chronic disease affecting seriously various aspects of fe-male health and wellbeing. It was shown to be associated with a significant increase of free circulating DNA (fcDNA) and gene methylation profile changes. The purpose of this study was to evaluate the effect of deoxyribonuclease (DNase) I therapy on these anomalies. Methods: fcDNA extraction, quantification and methylation status were performed with the use of commercial kits. DNase I was administered subcutaneously every 2 days for one month, and the pre-treatment and post-treatment fcDNA levels and methylation sta-tus, along with the degree of patients’ discomfort, were compared. Results: DNase I treatment decreased the overall level of fcDNA. Moreover, the treatment re-sulted in a significant change in methylation status (in the sense of hypermethylation or hypomethylation) in 6 out of the 9 genes targeted. Moreover, most of the patients (15/16) reported significant reduction in pain, an improvement in their ability to as-sume professional activity and less difficult sexuality. Conclusions: This is the first study reporting that DNase I treatment decreased the level of fcDNA, it did change the methylation status of some gene and, more importantly, it alleviated the clinical burden of the disease. Further research into the potential use of DNase-based medicine for endometriosis patients, including more participants, is warranted.

Abstract: Water is essential for human life, and access to clean water is considered a basic human right by the United Nations. Around the world, a high proportion of the population still does not have access to safe fresh water, with a high impact on health. This situation perpetuates a cycle of poverty, hindering economic development and exacerbating inequality. The water is considered unsafe to drink if it is contaminated. The contamination can be categorized into three types: physical, chemical, and biological. Biological contamination arises from the presence of living organisms in water, such as bacteria, viruses, algae, fungi, and parasites. Recently, the scientific community has raised the alarm on contamination caused by a large group of bacteria known as Cyanobacteria, which can release harmful toxins in water, including mycotoxins like Microcystin-LR (MC-LR). In this context, we present the application of fluorescence correlation spectroscopy (FCS) to develop a competitive assay for detecting the presence of traces of the MC-LR toxin in fresh water.

Abstract: Replication stress (RS) is a primary driver of genomic instability in cancer, yet the contribution of transcription-coupled repair to this process remains poorly understood. Here, we investigate how the TC-NER factor ERCC6 (CSB) shapes mutational landscapes under RS. We demonstrate that ERCC6 deficiency impairs replication restart and biases early damage signaling toward a 53BP1-mediated response, ultimately leading to senescence. Conversely, ERCC6-proficient cells prioritize survival and proliferative recovery but at the cost of distinct genomic alterations. Whole-exome sequencing reveals that ERCC6 drives the retention of stress-induced mutations specifically within coding regions of transcriptionally active loci, whereas ERCC6-deficient cells accumulate variants primarily in intergenic regions. These findings uncover a survival-mutagenesis trade-off: ERCC6 safeguards transcriptional continuity during replication stress but promotes mutational burdens in functional genomes. This mechanism parallels bacterial adaptive mutagenesis, identifying ERCC6 as a context-dependent driver of somatic evolution and tumor heterogeneity.

Abstract: X-ray crystallography remains the gold standard for high-resolution structural biology, yet obtaining diffraction-quality crystals continues to pose a major bottleneck due to inherently low success rates. This review advocates a paradigm shift from probabilistic screening to rational engineering, reframing crystallization as a controllable self-assembly process. We provide a comprehensive overview of strategies that connect fundamental physicochemical principles to practical applications, beginning with contact design, which involves the active engineering of crystal contacts through surface entropy reduction (SER), introduction of electrostatic patches, and strategic Lys-to-Arg substitutions to strengthen electrostatic interactions and improve enthalpic favorability. We also address scaffold design, utilizing rigid fusion partners and polymer-forming chaperones to promote crystallization even from low-concentration solutions. Furthermore, we highlight principles for controlling the behavior of multi-component complexes, based on our experimental experience. Finally, we examine de novo lattice design, which leverages AI tools such as AlphaFold and RFdiffusion to program crystal lattices from first principles. Together, these strategies establish an integrated workflow that links thermodynamic stability with crystallizability.

Abstract: The rapid proliferation of AI/ML models in drug discovery heralds an era of extraordinary progress, but also raises urgent questions about whether the true predictive performance is as good as advertised. On-target prediction models often benefit from high-resolution structural or atomistic representations that capture the subtleties of binding affinity and pose. By contrast, off-target and ADMET liabilities have typically relied on more implicit representations of molecular interactions. Retrospective benchmarks often provide a misleading picture of how successful these diverse representations are at predicting properties, and the community lacks standardized, prospective comparisons. Blind challenges, such as the OpenADMET × ASAP × PolarisHub Challenge featured in this issue, are crucial for realistically evaluating progress, encouraging iterations, and directing collective efforts toward major accuracy barriers. With ongoing investment in large-scale, open data creation and community-led challenges, predictive modeling is poised to rapidly transform drug discovery by enabling accurate, multi-parameter optimization.

Abstract: Obesity is a systemic metabolic disorder that is known to impair various organ systems; however, its precise impact on salivary gland homeostasis remains unclear. Recent studies have implicated ferroptosis—an iron-dependent form of regulated cell death characterized by lipid peroxidation and oxidative stress—in glandular dysfunction. In this study, we used leptin-deficient (ob/ob) mice to elucidate the role of ferroptosis in obesity-associated salivary gland pathology. The protective effects of ferroptosis in-hibition were evaluated by administering ferrostatin-1 (a lipid reactive oxygen species [ROS] scavenger) and deferoxamine (an iron chelator) for an 8-week period. Obese mice exhibited significantly increased body weight, food intake, and hyperglycemia. These systemic changes are accompanied by profound histological alterations in the salivary glands, including lipid droplet ac-cumulation, acinar atrophy, and mitochondrial ultrastructural damage. These alterations correlate with the hallmarks of ferrop-totic injury, including increased ROS levels, elevated malondialdehyde levels, suppressed glutathione peroxidase 4 activity, and iron overload. Salivary gland fibrosis, inflammation, and secretory dysfunction were evident, characterized by the upregulation of TGF-β and Collagen I, reduced expression of aquaporin-5 and amylase, and dysregulated levels of autophagy-related markers (LC3B and p62). Treatment with either ferrostatin-1 or deferoxamine significantly mitigated these pathologies; however, the degree of efficacy varied depending on the specific parameters that were examined. Thus, our findings implicate ferroptosis is a critical contributor to salivary gland dysfunction in obesity, and suggest that pharmacological inhibition of this pathway repre-sents a viable therapeutic strategy for preserving glandular integrity under metabolic stress.

Abstract: Cervical cancer remains a prominent cause of cancer‑related mortality among women worldwide because of chronic infection with high‑risk human papillomavirus (HPV) and disparate access to prevention and treatment. The current research evaluates the anticancer activity of Gypenoside XVII, a bioactive saponin of Gynostemma pentaphyllum, in HeLa cells as a model of cervical cancer. MTT, Annexin V-PI, and Hoechst 33342 assays showed dose‑dependent growth inhibition with typical apoptotic morphology. Flow cytometry revealed G₀/G₁ cell‑cycle arrest, while pathway interrogation revealed participation of mitochondrial and death‑receptor cascades, in agreement with caspase‑9 and caspase‑8 activation, respectively. Collectively, these findings position Gypenoside XVII as a natural‑product bioactive with potential both as an anticancer lead and as a functional‑food ingredient, deserving of further preclinical development.

Abstract: Background/Objectives: Post-COVID-19 pulmonary fibrosis (PCPF) and idiopathic pul-monary fibrosis (IPF) show clinical parallels, suggesting overlapping pathogenesis. This study investigated the dysregulation of key proteases, matrix metalloproteinases-2 and -9 (MMP-2/9), and associated inflammatory and endothelial markers in both conditions. Methods: We analyzed MMP-2 and MMP-9 gene expression in peripheral blood leuko-cytes and corresponding plasma protein levels in patients 6 and 12 months after SARS-CoV-2 infection, stratified by the presence (FB+) or absence (FB-) of post-COVID pulmonary fibrosis. These groups were compared to IPF patients and pre-pandemic healthy controls. Results: Results showed a significant, sustained increase in MMP-2/9 in post-COVID patients versus controls, which was most pronounced in the PCPF group and mirrored the dysregulation in IPF. This proteolytic shift corresponded to a distinct sys-temic profile: patients without fibrosis showed reduced levels of acute-phase cytokines (TNF-α, IL-6), whereas patients with fibrosis exhibited both elevated cytokines and in-creased markers of endothelial dysfunction (Endothelin-1, sICAM-1). Conclusions: The findings demonstrate that sustained MMP-2/9 overexpression is a hallmark of post-COVID fibrosis and is associated with a transition from systemic inflammation to chronic endothelial impairment. The convergence of this molecular profile in PCPF and IPF indicates shared pathophysiological pathways driving fibrosis. This positions MMP-2 and MMP-9 as promising biomarkers and potential therapeutic targets for mitigating progressive fibrotic lung disease.

Abstract: Faithful DNA replication is essential for genome stability but is constantly challenged by metabolic and oxidative stresses. Hydroxyurea (HU), a widely used antiproliferative drug, is traditionally known to inhibit ribonucleotide reductase and deplete dNTP pools. Recent studies, especially in Saccharomyces cerevisiae, reveal that HU-induced replication stress also arises from reactive oxygen species (ROS), which oxidize DNA, impair iron–sulfur–dependent replication enzymes, and disrupt replisome function. These combined effects promote helicase–polymerase uncoupling, accumulation of RPA-coated ssDNA, and activation of the Mec1–Rad53 (ATR–CHK1) checkpoint, leading to strand-specific changes such as PCNA unloading and reduced lagging-strand synthesis. When protective pathways are overwhelmed, HU-treated forks collapse, generating chromosome breaks and genome instability. This review summarizes current understanding of how HU remodels replication forks through both ROS-dependent and ROS-independent pathways and highlights emerging insights into how these mechanisms influence genome stability and may be exploited for therapeutic benefit.

Abstract: INTRODUCTION: While the exact cause of Type 1 Diabetes (T1D) remains unclear, it is widely believed that both genetic and environmental factors contribute to the development of the disease. Recent research has explored the potential role of gut microbiota and its metabolites in modulating immune responses and influencing the development of autoimmune diseases like T1D. With this purpose, we designed a study: 1. to compare the levels of different bacterial metabolites in plasma samples of T1D patients and healthy controls (HC). 2. to correlate the levels of these metabolites with different demographic, clinical and analytical variables collected from the T1D patients. METHODS: A total of 91 T1D patients were recruited. Plasma samples were collected and analyzed with gas chromatography coupled to mass spectrometry for the detection of the metabolites: short-chain fatty acids (SCFA: acetate [AA], propionate [PA], isobutyrate [IBA], butyrate [BA], isovalerate [IVA], valerate [VA] and methyl valerate [MVA]), medium-chain fatty acids (MCFA: hexanoate [HxA] and heptanoate [HpA]) and para-cresol (p-cresol). We also calculated the ratios between the different SCFA with AA. RESULTS: 1. AA levels were significantly higher in T1D patients than in HC (p=0.0009). PA/AA and IBA/AA ratios were significantly higher in HC (p=0.0004 and p=0.0001, respectively). 2. Glycated haemoglobin (HbA1c) was positively correlated with AA levels (p=0.0001; r=0.406) and a significant negative correlation with a rSpearman< -0.3 was found for PA/AA, IBA/AA and BA/AA ratios. 3. p-cresol correlated with Ferritin levels (p=0.04; r=0.362); besides, p-cresol levels were lower in T1D patients with a normal liver profile (p=0.002) and in T1D patients without hypertension (p=0.005). CONCLUSIONS: Serum levels of bacterial metabolites were significantly different in T1D patients. AA levels were significantly increased in T1D patients and p-cresol was higher in T1D patients with liver disturbances and hypertension. To develop strategies to restore gut microbiota health and immune balance could be essential for the control of T1D.

Abstract: The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag enhancing solubility of recombinant proteins and enabling purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF-mCherry accumulated predominantly in the in-soluble fraction, with only traces detectable in soluble lysates. Soluble CusF-mCherry did not bind Cu²⁺-charged IMAC resin, while moderate binding to Ni²⁺-charged resin was attributable to the vector-encoded His-tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not always bind Cu-based IMAC resin. Our findings expand empirical knowledge on solubility tag per-formance and emphasize the necessity of testing multiple tags to identify optimal strat-egies for recombinant protein production.

Abstract:

RNA chaperones play a crucial role in the biogenesis and function of various RNAs in bacteria. They facilitate the interaction of small regulatory trans-encoded sRNAs with mRNAs, thereby significantly altering the pattern of gene expression in cells. This allows bacteria responses quickly to changing environmental conditions, such as stress or adaptation to host organisms. Despite the identification of a large number of sRNAs in mycobacteria, none of the most common RNA chaperones has been found in their genomes. We characterized the cold shock protein CspB from Mycobacterium tuberculosis as a potential RNA chaperone. It forms a dimer due to its elongated C-terminal region, which is a hairpin composed of two α-helices. We demonstrated that CspB from M. tuberculosis exhibits high affinity for the two studied sRNAs from the same organism and, unlike the single-domain CspA, could be considered as a potential RNA chaperone in mycobacteria. Thus, it may be involved in the regulation of bacterial pathogenesis via interactions with sRNAs.

Abstract: Photosynthesis, a crucial component of the global carbon cycle, and biological nitrogen fixation (BNF), a key step in the nitrogen cycle, are both requisite for maintaining life and sustainable agriculture on Earth, in which chlorophyll and nitrogenase play central roles, respectively. In chlorophyll biosynthesis, the reduction of protochlorophyllide to chlorophyllide is catalyzed by either the nitrogenase-like, light-independent DPOR (dark-operative protochlorophyllide oxidoreductase) or the SDR-like, light-dependent LPOR (light-driven protochlorophyllide oxidoreductase), utilizing chemical and light energy. Nitrogenase (N₂ase) is the only enzyme capable of reducing inert atmospheric nitrogen, yet only a few bacterial species possess the multi-subunit ATP-dependent dinitrogen reductase. Over the past three decades, significant progress has been made in solving the crystal structures of N₂ase, DPOR, and LPOR protein complex, providing valuable mechanistic insights into N₂ase, as well as the contrasting structures of the multi-subunit DPOR and single-subunit LPOR. We summarize the structural breakthroughs of these key catalysts for nitrogen and carbon fixation. Protein structural similarities often hint at similar functions and evolutionary relationships. With the ongoing development in protein structure annotation assisted by AlphaFold and the identification of protein structure similarities through structural alignment software, the evolutionary relationship between these key enzymes in photosynthesis and nitrogen fixation, as well as the potential co-origin of these processes, has been uncovered. We further summarize how a porphyrin reduction catalyst evolves from a less efficient ATP-driven DPOR to a highly efficient light-driven LPOR. The key structural milestones achieved in N₂ase, DPOR, and LPOR research laid the groundwork for future perspectives, particularly in proposing a direction for a potential nitrogen-fixing scenario using an AI-designed LUN and photosynthesis with bacteriochlorophyll using a light-driven chlide reductase (LCOR), leading to the development of more effective forms of biological nitrogen fixation and photosynthesis.

Abstract: During ischemia, endothelial cells' integrity is compromised, as a consequence, blood–barrier homeostasis is disrupted. Therefore, the structural and functional preserva-tion of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightfor-ward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 hours of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hy-poxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. The mod-el enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress reso-lution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to its mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approach-es that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress–modulating interven-tions aiming at preserving endothelial barrier function, in various ischemic contexts.