Frontiers in Molecular Biosciences

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

Bis(monoacylglycero)phosphate (BMP) is a signature lysosomal phospholipid that supports the catabolic functions of the lysosome. We recently demonstrated that BMP deficiency is associated with a variant of Batten disease, a neurodegenerative lysosomal storage disorder. This observation led us to investigate how BMP deficiency contributes to the previously reported accumulation of the ATP synthase c-ring in the lysosomes of Batten disease patients and preclinical models. The c-ring is an inner mitochondrial membrane (IMM) protein complex that interacts with cardiolipin, a mitochondrial phospholipid that shares structural features with BMP. Based on this, we hypothesised that BMP may perform an analogous function to cardiolipin in lysosomes. Specifically, we proposed that BMP preferentially interacts with the c-ring, dispersing it within lysosomal membranes and facilitating its degradation. To test this hypothesis, we conducted all-atom molecular dynamics simulations to examine the interactions of various BMP variants with the c-ring of human ATP synthase under different membrane conditions. We observed leaflet-specific preferential interactions of BMP with the protein interface. Replacement of BMP with anionic POPG lipids resulted in a lower binding affinity to the c-ring, indicating the importance of BMPs unique structure with respect to protein binding. Furthermore, BMP enrichment was enhanced when using the physiologically relevant di-22:6 BMP variant in membranes containing polyunsaturated lipids and cholesterol. Overall, our study suggests that BMP promotes lysosomal c-ring degradation via c-ring co-localisation, whereas BMP deficiency in Batten disease drives c-ring accumulation. Significance statementBatten disease comprises a group of neurodegenerative lysosomal storage disorders that primarily affects infants and children. Several recently identified disease genes are linked to the biosynthesis of lipid bis(monoacylglycero)phosphate (BMP), a lysosomal lipid required for normal lysosome function. However, how BMP deficiency contributes to disease pathology remains poorly understood. Here, we investigate the relationship between BMP deficiency and lysosomal accumulation of the ATP synthase c-ring, a hallmark pathological feature of Batten disease. Using molecular simulations, we show that BMP preferentially interacts with the c-ring, suggesting that BMP deficiency promotes c-ring aggregation within lysosomes. Our study provides a mechanistic starting point for understanding how BMP deficiency disrupts lysosomal function in Batten disease

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

1A-adrenoceptor (1A-AR) is a class A G-protein coupled receptor (GPCR) that stimulates smooth muscle contraction in response to adrenaline and noradrenaline. GPCRs exist in a dynamic equilibrium between multiple conformational states. Ligand binding induces structural rearrangements via conserved microswitches, which are thought to shift the equilibrium and trigger signalling. For structural and biochemical studies, GPCRs must be solubilised from the membrane, typically using detergent micelles. However, detergents can disrupt native dynamics of membrane proteins, potentially confounding experimental results. To address this, phospholipid bilayer mimetics such as nanodiscs and saposin nanoparticles (SNPs) have been developed to provide a more native-like environment. Thermostabilised 1A-AR serves as a GPCR prototype and can be expressed and isotopically labelled for NMR purposes. To investigate how membrane mimetics influence the conformational diversity of 1A-AR, we compared 1H 13C3-HMQC NMR experiments of 13CH3-Met labelled 1A-AR incorporated into either DDM, LMNG, or SNPs, in presence of ligands with varying efficacies. Several methionine residues are positioned near key microswitches, including M2035.57, located closed to the G protein binding site. Its resonance has been proposed as a readout of receptor conformational state, shifting with ligand efficacy. Spectra of 13CH3-Met labelled 1A-AR in LMNG closely resembled those in DDM with some temperature-dependent dynamic variation. In contrast, incorporation into SNPs led to a complete loss of M2035.57 signal, consistent with an intermediate exchange rate. These findings demonstrate that the membrane environment can profoundly influence conformational dynamics in GPCR NMR studies. Our results highlight the need to carefully consider membrane environment when interpreting NMR data and underscore the value of benchmarking against biologically relevant controls.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

Background: Olfactory dysfunction is a recognised but poorly characterised comorbidity of Primary Ciliary Dyskinesia (PCD). No prior systematic review has synthesised its prevalence or clinical correlates. Methodology: A PRISMA compliant systematic review and meta-analysis was conducted. Five databases were searched to February 2026. Observational studies reporting olfactory function in confirmed PCD were included. Risk of Bias was assessed using the Newcastle-Ottawa Scale. A random-effects meta-analysis using the Freeman-Tukey double arcsine transformation was performed to calculate pooled prevalence with 95% confidence intervals (CI) and prediction intervals (PI). Results: Twelve studies (n=865) were included. Overall pooled prevalence of olfactory dysfunction was 43.4% (95% CI 25.2-62.5%; 95% PI 0.1-99.0%). Objective psychophysical testing yielded a significantly higher pooled prevalence of 66.1% (95% CI 55.5-76.0%; 95% PI 38.4-88.9%) compared to patient-reported outcome measures (30.5%; 95% CI 11.4-54.0%). Older age, greater sinonasal disease burden, and specific ciliary ultrastructural defects were associated with worse olfactory function. A striking discordance between objective dysfunction and subjective awareness was observed across multiple studies. Conclusions: Olfactory dysfunction is highly prevalent in PCD and substantially under-recognised by patients. Routine objective olfactory screening should be integrated into standard multidisciplinary PCD care.

Background/Objectives. Autism spectrum disorder (ASD) has been associated with microbiome alterations, but the relative contribution of environmental and individual factors remains unclear. This study explored oral and gut microbiome profiles in environmentally matched dizygotic triplets discordant for ASD. Materials and Methods. Triplets in the 5-9 year age range, including one child with ASD and two neurotypical siblings, underwent standardized oral examination. Oral tongue-dorsum and rectal swab samples were analyzed by 16S rRNA sequencing. Taxonomic composition and beta diversity were evaluated descriptively. Results. Dominant bacterial phyla were broadly similar across siblings, but oral microbial profiles showed greater interindividual variation. The participant with ASD had the highest dental biofilm accumulation, predominance of Streptococcus, and reduced representation of several secondary genera. One neurotypical sibling with mild gingival inflammation showed greater representation of Fusobacterium, Prevotella, and Leptotrichia. Beta diversity demonstrated clearer interindividual separation among oral than gut samples. Conclusions. Individual-specific factors may influence microbiome patterns even under highly similar environmental and dietary conditions. These findings support further investigation of the oral microbiome as a complementary component of ASD microbiome research.

Background: Comparisons between independently predicted wild-type and missense-variant protein structures can generate mechanistic hypotheses, but small apparent differences may reflect model-selection variability rather than mutation-specific effects. Methods: Human mitochondrial DNA polymerase gamma (POLG; UniProt P54098) variants p.Arg627Gln (R627Q) and p.Trp748Ser (W748S) were evaluated using five AlphaFold2-PTM network-model outputs per condition generated with one random seed under matched ColabFold settings. Ten pairwise wild type comparisons at each site described between-network model-selection variability. Variant effects were summarized across five within-network wild-type-versus-variant comparisons using rotation-invariant local C-alpha pair distances and local displacement after global and local alignment. Because these comparison designs differ, the wild-type distribution was used as context rather than a mutation-effect null. Wild-type cryo-EM structure 9GGF was used for contact and interface mapping. Experimental A467T and G848S structures 9GGE and 9GGC provided contextual benchmarks. Results: R627Q measurements fell within the range of between-network wild-type differences: its median mean local pair-distance change was 0.170 angstrom, compared with a wild-type median of 0.170 angstrom, and its locally aligned displacement was 0.265 versus 0.248 angstrom. W748S showed higher median values (0.168 versus 0.132 angstrom for pair-distance change; 0.236 versus 0.182 angstrom for locally aligned displacement), but the ranges overlapped and the comparison-design asymmetry precluded a calibrated mutation-effect percentile. Experimental A467T and G848S comparisons produced local changes of similar magnitude. In 9GGF, R627 and W748 directly shared a local microenvironment, with a minimum heavy-atom distance of 3.53 angstrom. R627 also formed short polar-contact candidates with D629 and D743, whereas W748 occupied a hydrophobic packing environment containing Y622 and F750. Both sites were more than 18 angstrom from nucleic acid, more than 30 angstrom from POLG2, and more than 33 angstrom from PZL-A in a ligand-bound structure. Conclusions: Available AlphaFold2 comparisons do not establish a mutation-specific structural deformation for either variant. Experimental-structure mapping supports testable physicochemical hypotheses involving a shared R627-W748 microenvironment - loss of an arginine-centered polar network for R627Q and disruption of a buried aromatic environment for W748S - but not direct DNA, POLG2, or PZL-A contact mechanisms. Matched control substitutions and independent seeds are required to calibrate small mutation-associated structural deltas.

Imipramine is known as an older generation tricyclic antidepressant drug. It has been identified in prior studies that imipramine blocks Apolipoprotein E4 (ApoE4)-induced amyloid-{beta}(A{beta}) aggregation and is associated with an improved AD diagnosis [Johnson et al. Alzheimers Research Therapy, 2022, 14, 88]. Using NMR methods such as 1H-1H NOESY and Saturation Transfer Difference Spectroscopy, we demonstrate the binding of A{beta} monomers to imipramine when the full-length A{beta} (1-42) sequence is considered. The more abundant but less toxic form, A{beta} (1-40) does not show interaction with imipramine.

Solution NMR spectroscopy provides atomistic measurements of proteins in a native-like biophysical state. Because these measurements are ensemble averages, it also has the potential to report on conformational diversity. However, conventional NMR structure determination typically converts experimental observables into restraints for molecular dynamics, which encode information on the mean structure but do not retain information on the underlying conformational distribution. Ensemble selection has long been proposed as an alternative, whereby experimental observables are compared directly with candidate conformers generated independently of the measurements. This allows population distributions to be inferred from the data. However, few such methods have incorporated NOESY - the richest source of structural information in protein NMR - data, due to challenges in the quantitative comparison of experimental and back-calculated spectra. To address this challenge, we previously introduced the CoMAND method, demonstrating that quantitative agreement is practical for NOESY spectra with bespoke heteronuclear editing schemes. Here we extend this approach into a framework for direct inference of protein ensembles within a flexible ensemble-selection architecture incorporating multiple classes of NMR observables. We introduce a quantitative scoring framework for comparing experimental and back-calculated observables and combine it with regularized ensemble selection and Monte Carlo simulated annealing. Integration with the OpenMM molecular dynamics engine allows conformational pools to be generated using established molecular simulation methods. Applied to human ubiquitin, the resulting ensemble provides simultaneous agreement with NOESY, residual dipolar coupling and scalar coupling data while retaining conformational diversity supported by experiment.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Interleukin-13 (IL-13) is an immunomodulatory cell signaling cytokine that has been implicated in neurodegenerative disease and chronic inflammation. IL-13 binds to its low and high affinity receptors, IL-13 receptor 1 (IL-13R1) and IL-13 receptor 2 (IL-13R2), respectively, with residence times that vary accordingly. As the binding kinetics of the cytokine-receptor complexes influence cellular responses, we employed the molecular dynamics (MD) simulation-based{tau} -random acceleration molecular dynamics method ({tau}RAMD) to compute relative residence times for wild-type (WT) IL-13 and 19 IL-13 mutants to the two receptors. Comparison with experimental kinetic data shows that the{tau} RAMD computations capture the trends in residence times. Analysis of simulated dissociation trajectories of the cytokine-receptor complexes reveals two distinct dissociation pathways of IL-13 from each of the receptors. This study thus pinpoints key determinants of the interaction of IL-13 with its receptors which could be targeted for therapeutic design. Statement of SignificanceCytokines are regulatory proteins that bind to cell surface receptors and thereby send signals to the cellular interior. Interleukin-13 (IL-13) is a cytokine that has a low and a high affinity receptor. It has important physiological roles, and its deregulation is involved in diseases such as atopic dermatitis and asthma. We employed a molecular dynamics simulation-based method to compute the effects of changes in the sequence of IL-13 on the lifetimes of complexes of IL-13 and its receptors. Comparison with experiments supports the validity of the computational approach and analysis of the simulations reveals two distinct ways in which IL-13 dissociates from each receptor. These results thus provide a map for targeting IL-13 - receptor interactions for the design of therapeutics.

Primary ciliary dyskinesia (PCD) is a rare, genetic, multiorgan disease requiring lifelong management. Although PCD affects everyday life, little is known about how people with PCD experience social functioning (SF). We conducted a study within the international participatory Living with PCD study to comprehensively explore SF. First, we conducted a focus group and two semi-structured interviews with adults and parents of people with PCD. We analysed qualitative data thematically and used the findings to develop a multilingual online questionnaire on SF. The questionnaire was completed by 277 participants: 225 adults and adolescents with PCD (81%) and 52 parents of children with PCD (19%). Participants reported active social lives and strong close relationships. PCD had a positive impact on family relationships for 39% of adult/adolescent participants and 41% of parents reporting for children. Among adult/adolescent participants, 49% reported positive or no impact on romantic/intimate relationships, while 17% had avoided or ended a relationship because of PCD. PCD affected the ability to meet responsibilities for 54% of participants, free time for 58%, and planning effort for 53%. Participants were more comfortable discussing PCD with family, friends, and partners than in work or educational settings, where only 29% reported receiving support. Financial support, flexible work, or educational policies and better-trained healthcare professionals were the most frequently identified unmet needs. This study suggests that maintaining SF with PCD requires substantial individual and relational work. Improving SF for people with PCD requires systemic responses in healthcare, education, and employment, alongside support from close networks.

The gut microbiome maintains a complex, bidirectional communication network with the central nervous system, commonly referred to as the gut-brain axis and its disruption has been implicated in several neurological disorders. This study combines a survey-based assessment of public awareness with an in-silico comparative analysis of gut microbial dysbiosis across four prevalent neurological disorders as observed in the current study: depression, anxiety, schizophrenia and autism spectrum disorder (ASD). A structured, anonymous online survey (n = 230) captured perceptions of the gut-brain connection along with dietary, lifestyle and gastrointestinal correlates of stress in a predominantly young, health-sciences-affiliated Indian cohort. In parallel, disorder-specific lists of elevated and reduced faecal microbial taxa were retrieved from the Disbiome database, compared using a multiple list comparator and taxonomically classified using the NCBI Taxonomy tool to construct phylogenetic trees in iTOL. Approximately three-quarters of respondents were aware of a potential gut-mental health link, yet about half reported no specific dietary practice and roughly 60% experienced stress-related digestive symptoms while rarely seeking medical consultation for them. Comparative analysis showed that depression, anxiety and schizophrenia shared a substantially overlapping dysbiosis signature, with common elevation of Actinomyces, Bacteroidaceae, Blautia, Eggerthella, Oscillibacter, Parasutterella and Veillonella and common reduction of Coprococcus, Lachnospiraceae, Ruminococcaceae, Clostridium, Faecalibacterium and Sutterella. In contrast, ASD displayed a distinct microbial signature with limited overlap with the other three disorders. Phylogenetic clustering confirmed that the shared taxa belonged predominantly to the phyla Bacillota (formerly Firmicutes), Bacteroidota (formerly Bacteroidetes), Actinomycetota (formerly Actinobacteria) and Pseudomonadota (formerly Proteobacteria). Notably, this phylum-level pattern parallels recent comparative analyses of microbial dysbiosis in neurodegenerative diseases, suggesting that broad phylogenetic shifts may be a relatively general correlate of chronic neurological disease, while disorder specificity emerges at the level of individual taxa. These findings support a shared microbial pathway linking depression, anxiety and schizophrenia that is distinct from the dysbiosis pattern observed in ASD and they underscore the value of microbiome-informed, disorder-specific therapeutic strategies.

Objectives: To test whether radiology report evaluation metrics distinguish clinically meaningful errors from textual changes and align with radiologist-assessed error burden. Methods: Cross-dataset evaluation used ReXErr-v1 (2,708 report pairs; 5,724 paired error sentences) and 100 RadEvalX report pairs with expert error counts. BLEU-4, ROUGE-L and METEOR were assessed in ReXErr-v1; RadEvalX analyses included these plus BERTScore, CheXbert, RadGraph F1 and RadCliQ. Outcomes were ReXErr-v1 pairwise win rate and AUROC for clinical-content versus linguistic errors, and RadEvalX Spearman correlation with clinically significant error count and AUROC for any significant error. Confidence intervals used 10,000 clustered percentile bootstrap resamples; Holm adjustment-controlled multiplicity. Results: ReXErr-v1 paired-sentence win rates were 0.986 for BLEU-4, 0.999 for ROUGE-L and 0.998 for METEOR, but discrimination of clinical-content from linguistic errors was modest (AUROC 0.609-0.620). Penalty magnitude was strongly associated with textual change after adjustment for error type (normalised character edit distance coefficient 0.746; 95% CI 0.705-0.788; P<0.001). In RadEvalX, CheXbert showed the highest correlation with clinically significant errors (rho=0.413; 95% CI 0.223-0.578) and highest AUROC (0.742; 95% CI 0.638-0.836). Conclusions: Near-ceiling sensitivity to textual corruption did not imply sensitivity to clinical significance. CheXbert showed the highest alignment with expert error assessment, although pairwise superiority was not demonstrated over all comparators and performance remained moderate.

ObjectiveThe complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. MethodsThe interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM [&ge;] 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results(1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. ConclusionCD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting that these sites may tolerate substitutions or that redundant contact residues maintain the interaction.

Antibodies generally exhibit high specificity for their cognate epitopes, but structural and physicochemical similarities between distinct epitopes can enable an antibody to recognize different antigens, resulting in cross-reactivity. This property can be exploited for antibody repurposing. To identify epitopes that share such similarities, both sequence- and structure-based approaches can be employed. In this context, 3D Zernike descriptors provide a compact representation of protein surface geometry as numerical feature vectors, enabling quantitative comparisons independently of structural alignment and orientation. Thus, this study aimed to evaluate the application of 3D Zernike descriptors for the structural clustering of antibodies and epitopes and to explore their use in antibody repurposing for the recognition of new targets. To this end, antibody binding sites previously associated with recognition of similar epitopes were analyzed at different structural levels, considering the CDRs, CDRH3, and complete paratopes. Surface similarity was subsequently quantified by calculating the Euclidean distance between their corresponding 3D Zernike feature vectors. Performance was benchmarked against SPACE2. Additionally, different distance thresholds were evaluated based on their ability to recover antibody pairs recognizing the same epitope. The paratope-based approach provided the best balance between the number of identified pairs and precision at a distance threshold of 2.7, whereas epitope clustering showed robust performance up to a distance of 3.0. At these thresholds, the 3D Zernike descriptors identified a greater number of functional pairs than SPACE2 while maintaining comparable precision and identifying complementary sets of antibody pairs.. BTaken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing G, a highly lethal zoonotic pathogen. Structural screening identified three antibodies with epitopes similar to the NiV target that also showed a consistent binding preference for the target epitope in molecular docking assays. Notably, one candidate, originally directed against a SARS-CoV-2 epitope, formed a stable complex with the NiV epitope, remaining within the 5 [A] RMSD threshold during heated molecular dynamics simulations and emerging as a potential cross-reactive candidate.These results support the use of this computational framework for biopharmaceutical discovery against emerging targets. Taken together, these findings support the use of 3D Zernike descriptors for structural clustering of antibodies and epitopes and for guiding antibody repurposing.