Search Results (114)

Papain-like protease (PLpro), a crucial functional domain of the SARS-CoV-2 non-structural protein 3 (nsp3), plays a dual role in both hydrolyzing viral polyprotein precursors and modulating host immune responses. These critical functions position PLpro as a key target in the ongoing development of antiviral therapies for SARS-CoV-2. This review analyzes more than 100 PLpro-ligand co-crystal structures and summarizes the major binding modes between these ligands and PLpro. Most of these ligands bind to sites analogous to those targeted by the classical non-covalent inhibitor GRL0617, primarily involving the P3 and P4 subsites and the BL2 loop. Based on these structural insights, optimized inhibitors have expanded targeting beyond the canonical binding site to auxiliary regions such as the BL2 groove and the Val70 site, and in some cases toward the catalytic Cys111 buried within a narrow pocket. Certain ligands identified through various screening approaches bind to non-canonical or allosteric regions, such as the S1 and S2 sites or the zinc-finger domain, engaging PLpro through distinct interaction modes and thereby offering additional opportunities for PLpro inhibitor design. The review also discusses potential strategies for future PLpro inhibitor development informed by recent structural advances. Taken together, these structural and functional insights support ongoing efforts in the structure-guided design and optimization of PLpro inhibitors. Full article

Background: Coffin–Siris syndrome 12 (CSS12) is a recently described neurodevelopmental disorder caused by heterozygous pathogenic variants in BICRA, a gene encoding a core subunit of the non-canonical BAF (ncBAF) chromatin-remodeling complex. The condition is characterized by developmental delay, hypotonia, hypertrichosis, and joint laxity. However, long-term data remain limited, and systemic manifestations are incompletely defined. Case Description: We report a 22-year-old male with a de novo BICRA frameshift variant, c.2479_2480delinsA (p.Ala827Thrfs*15), previously included in the original cohort reported by Barish et al. Longitudinal follow-up revealed an expanded phenotype extending beyond neurodevelopmental features. Early findings included global developmental delay, growth hormone deficiency, short stature, and joint hypermobility. In adolescence and adulthood, he developed severe intestinal dysmotility requiring total colectomy, recurrent spontaneous pneumothoraces from bilateral apical bullous disease, and portal-vein thrombosis, representing visceral and vascular complications not previously emphasized in BICRA-related disorders. The identified BICRA variant truncates the coiled-coil domain critical for BRD9/BRD4 interaction, consistent with a loss-of-function mechanism. The patient’s systemic features suggest that BICRA haploinsufficiency affects not only neurodevelopmental pathways but also smooth-muscle and connective-tissue integrity. Conclusions: This case expands the phenotypic spectrum of BICRA-related CSS12, demonstrating that visceral and vascular involvement can occur alongside neurodevelopmental and connective-tissue features. Recognition of these broader manifestations underscores the need for lifelong multidisciplinary surveillance and contributes to understanding the diverse biological roles of the ncBAF complex in human development. Full article

Background/Objectives: The 2021 WHO Classification of Central Nervous System (CNS) tumors emphasizes the integration of molecular data with histopathological features. Lower-grade gliomas (LGGs) represent a heterogeneous group of neoplasms with variable clinical behavior. This study aimed to explore the molecular landscape of a single-institution series of LGGs using targeted next-generation sequencing (NGS). Methods: Eleven adult patients diagnosed with LGG between 2015 and 2024 at Cattinara University Hospital (Trieste, Italy) were retrospectively analyzed. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and analyzed using the TruSight Oncology 500 panel (Illumina). Mutational, amplification, and transcriptomic profiles were evaluated. Results: IDH1 mutations were the most frequent alteration (75%), commonly co-occurring with TP53 and ATRX mutations, consistent with the canonical IDH-mutant astrocytoma profile. CDK4 amplification was found in four cases, while MYCN amplification and MET amplification were each identified in isolated cases. Two diffuse IDH-wild-type gliomas displayed aggressive clinical courses and shorter survival, and one was reclassified as glioblastoma (grade 4) based on EGFR amplification. The transcriptome analysis revealed heterogeneous expression signatures and distinct clustering of IDH1/ATRX-mutant tumors. Conclusions: Targeted NGS confirmed the key molecular features of diffuse gliomas and enabled precise WHO 2021 classification even in archival FFPE samples. Despite the exploratory nature of the analysis on a small population, the study underscores the biological and transcriptional heterogeneity of LGGs and highlights the limitations of tumor-only sequencing approaches. Broader genomic profiling and matched normal controls are warranted to refine the interpretation of rare or non-canonical variants. Full article

Background: Biliary tract cancer (BTC) is an aggressive malignancy with poor prognosis and limited therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated efficacy in tumors with homologous recombination repair (HRR) deficiency. However, actionable BRCA1/2 mutations are rare in BTC. Epigenetic modulation via DNA methyltransferase (DNMT) inhibition is a proposed strategy for inducing an HR-deficient (“BRCAness”) phenotype and thereby enhancing therapeutic response to PARP inhibitors. This study aimed to determine whether the DNMT inhibitor azacitidine (AZA) enhances the antitumor effects of the PARP inhibitor niraparib (NIR) and to identify molecular mechanisms underlying this interaction. Methods: Two BTC cell lines, TFK-1 and RBE, were treated with AZA and/or NIR or subjected to siRNA-mediated DNMT1, DNMT3A, or DNMT3B knockdown. Functional analyses included homologous recombination (HR) assays, flow cytometric evaluation of cell-cycle distribution and apoptosis, proliferation and survival assays, and IC₅₀ determination. Whole-transcriptome RNA sequencing was performed to identify differentially expressed genes after AZA treatment or DNMT3B knockdown, followed by validation via qPCR and Western blotting. To explore epigenetic regulation, whole-genome bisulfite sequencing was performed on TFK-1 cells following DNMT3B knockdown. Results: AZA treatment decreased HR frequency in a dose-dependent manner and enhanced the sensitivity of BTC cells to NIR, as evidenced by increased apoptosis, suppressed proliferation, and reduced IC₅₀ values. DNMT3B knockdown recapitulated these effects, establishing a causal relationship between DNMT3B suppression and disrupted HR repair. RNA sequencing identified opioid growth factor receptor (OGFR) as a commonly upregulated gene after DNMT3B knockdown. Functional validation showed that OGFR overexpression reduced HR activity, increased apoptosis, and enhanced NIR sensitivity. Contrarily, OGFR knockdown conferred relative resistance. Whole-genome bisulfite sequencing showed no significant CpG methylation changes at the OGFR promoter region, indicating that OGFR induction is mediated through DNMT3B-dependent transcriptional regulation rather than direct promoter demethylation. Conclusions: DNMT3B inhibition sensitizes BTC cells to PARP inhibitors by disrupting HR repair. OGFR was identified as a novel regulator of HR and PARP inhibitor sensitivity, controlled via noncanonical DNMT3B-dependent transcriptional mechanisms that operate independently of CpG methylation. These findings provide new mechanistic insights into the epigenetic control of DNA repair and support the rationale for combining DNMT and PARP inhibitors as a promising therapeutic strategy for BTC beyond genetically HR-deficient cases. Full article

LD-Transpeptidases (LDTs) are a widely conserved class of peptidoglycan (PG) crosslinking enzymes in bacteria. They are sometimes overlooked as they often act secondary to penicillin binding proteins (PBPs) under standard conditions. However, LDTs are essential in key pathogens such as Clostridioides difficile and are responsible for β-lactam resistance in Mycobacterium tuberculosis and Enterococcus faecium due their low affinity for penicillins and cephalosporins, allowing them to form LD-crosslinks when DD-crosslinking PBPs are inactivated. This role makes LDTs a promising target when developing new treatments for these pathogens. LDTs can perform different enzymatic reactions. Most commonly they reinforce the PG with 3,3-LD-crosslinks or, in a few cases, 1,3-LD-crosslinks, during stationary phase or stress responses. Some LDTs also incorporate endogenous and exogenous non-canonical D-amino acids into the PG. In many Gram-negative bacteria, specialised LDTs tether lipoproteins or outer membrane proteins (OMPs) to the PG to maintain cell envelope integrity; in some cases this regulates virulence factors. Specialised LDTs have also been implied to have roles in polar growth, toxin secretion, and symbiotic colonisation. Recent discoveries include novel subgroups of the major YkuD family and the identification of the VanW family; this has opened new research directions surrounding LDTs. We aim to understand LDTs and their roles to expand our knowledge of PG synthesis and modification and how these enzymes can be targeted for antibiotic treatment. Full article

SH3 domains are widespread protein modules that mostly bind to proline-rich short linear motifs (SLiMs). Most known SH3 domain-motif interactions and canonical or non-canonical recognition specificities are described for individual SH3 domains. Although cooperation and coordinated motif binding between tandem SH3 domains has already been described for members of the p47^phox-related protein family, individual cases have never been collected and analyzed collectively, which precluded the definition of the binding preferences and targeted discovery of further instances. Here, we apply an integrative approach that includes data collection, curation, bioinformatics analyses and state-of-the-art structure prediction methods to fill these gaps. A search of the human proteome with the sequence signatures of SH3 tandemization and follow-up structure analyses suggest that SH3 tandemization could be specific for this family. We define the optimal binding preference of tandemly arranged SH3 domains as [PAVIL]PPR[PR][^DE][^DE] and propose potential new instances of this SLiM among the family members and their binding partners. Structure predictions suggest the possibility of a novel, reverse binding mode for certain motif instances. In all, our comprehensive analysis of this unique SH3 binding mode enabled the identification of novel, interesting tandem SH3-binding motif candidates with potential therapeutic relevance. Full article

In this work, we adopted an approach similar to that of Chatzarakis’, by transforming the oscillation analysis of third-order differential equations into an equivalent first-order problem. A key generalization in our study is the extension coefficient $b\left(t\right)$ from the range $0\le b\left(t\right)\le 1$ to $b\left(t\right)\ge 1$. Moreover, we established several oscillation criteria applicable to the canonical and non-canonical cases. Our conclusions complement and extend the oscillation theory for third-order delay differential equations. Several examples are provided to illustrate our results. Full article

Mismatch repair (MMR) system alterations can trigger transient hypermutation, promoting adaptive mutations under stress, such as antibiotic exposure. While most organisms use MutS and MutL protein families for MMR, many archaea and actinobacteria, including the major human pathogen Mycobacterium tuberculosis, lack these components and instead rely on NucS, a structurally distinct enzyme driving a non-canonical MMR pathway. Given the role of MMR in mutation control, understanding how nucS expression is regulated could be essential for uncovering the molecular basis of antibiotic resistance development in mycobacteria. In this study, we characterized the nucS promoter and transcription start site in Mycobacterium smegmatis. We found that nucS expression declines during the stationary phase in both M. smegmatis and M. tuberculosis, paralleling replication activity and canonical MMR downregulation. Our data suggest that the alternative sigma factor σ^B may negatively regulate nucS expression during this phase. Additionally, we identified candidate compounds that may modulate nucS expression, underscoring its responsiveness to environmental cues. These findings enhance our understanding of mycobacterial stress responses and lay the groundwork for exploring antibiotic resistance mechanisms. Strikingly, our work reveals a case of double convergent evolution: both canonical (MutS/MutL) and non-canonical (NucS) pathways have independently evolved not only the same DNA repair function, but also similar regulatory frameworks for genome integrity preservation under stress conditions. Full article

Antimicrobial resistance (AMR) is traditionally discussed in the context of horizontally acquired resistance genes and point mutations at target loci. However, this gene-centred model fails to account for a large number of clinically important modalities of resistance. There is now substantial evidence implicating bacteria in the ability to escape the effects of antibiotics in a variety of non-canonical ways, which are not considered in traditional diagnostic and surveillance pipelines. Among these factors, we can list those arising from global regulatory networks, phase variability, epigenetic tuning, small RNAs, genome structural variability, and phenotypic states like tolerance and persistence. This review will blend the current knowledge on these alternative pathways of resistance and underscore how they intersect with canonical genetic determinants. We will highlight cases where resistance emerges in the absence of known resistance genes, analyse the role of regulatory plasticity in efflux pump expression and membrane remodelling, and examine the contributions of bacterial stress responses and post-transcriptional control. Additionally, we will address methodological gaps in the detection of these mechanisms and their implications for clinical treatment failure, resistance surveillance, and drug development. By integrating insights from molecular microbiology, systems biology, and genomics, this review aims to offer a framework for understanding AMR as a multifaceted, context-dependent phenotype, not merely a genotype. We conclude by identifying knowledge gaps and suggesting priorities for research and diagnostic innovation in this evolving field. Full article

Background/Objectives: Fluorescence in situ hybridization (FISH) is a standard diagnostic tool for detecting gene fusions and rearrangements in lymphomas but is limited by incomplete genomic coverage, dependence on predefined probes, and difficulty identifying atypical or noncanonical fusion partners. These constraints often result in inconclusive diagnoses in complex lymphoma cases. This study evaluates a novel Hi-C-based sequencing assay from formalin-fixed paraffin-embedded (FFPE) samples to detect clinically significant gene fusions and rearrangements in cases where conventional FISH was inconclusive or expected biomarkers were not detected. Methods: Five diffuse large B-cell lymphoma cases with previously atypical gene fusions or rearrangements by FISH were analyzed using both standard FISH and a Hi-C-based lymphoma assay. Standard FISH was performed using break-apart probes targeting MYC, BCL2, and BCL6, and dual-fusion probes targeting IGH::MYC and IGH::BCL2. The Hi-C assay utilized high-resolution sequencing of FFPE tissue to map chromatin interactions and identify structural variations across the genome and assessment of their clinical relevance. Results: In this series of five lymphoma cases, Hi-C detected additional structural variants beyond those identified by FISH. It identified typical and atypical translocation partners of key oncogenes (MYC, BCL2, BCL6), cryptic breakpoints, and novel genomic events, including TP53 loss, KMT2A amplification, and complex rearrangements, which were undetectable by FISH. The Hi-C assay’s whole-genome coverage enabled comprehensive profiling. Conclusions: The Hi-C-based lymphoma assay offers a transformative diagnostic tool, overcoming FISH limitations by providing unbiased, high-resolution detection of structural variations. This approach enhances diagnostic accuracy and supports personalized therapeutic strategies in lymphoma management, warranting further validation for clinical adoption. Full article

Background/Objectives: The Wnt signaling pathway is pivotal in the adenoma–carcinoma sequence; however, its role in small bowel adenocarcinoma (SBA) remains insufficiently characterized. We analyzed the clinicopathological significance of Wnt pathway-related gene mutations and the expression of downstream or associated proteins in SBA. Methods: Immunohistochemical staining for β-catenin, cyclin D1, c-Myc, E-cadherin, and Wnt5a was performed in 75 primary SBA surgical specimens. Targeted next-generation sequencing was conducted in 48 of these cases. Results: The genomic alterations in the Wnt pathway were identified as APC (14.6%) and CTNNB1 (8.3%), with no overlap between the two mutations. Aberrant (reduced membranous and/or nuclear) expression of β-catenin was observed in 37% of cases. Cyclin D1 and c-Myc were expressed in 60% and 41% of cases, respectively. Aberrant expression of β-catenin and/or Wnt5a was present in 60% of cases and was correlated with cyclin D1 and c-Myc expression. Mutations in APC and CTNNB1 were found in intestinal- and gastrointestinal-type SBAs, but were absent in gastric-type SBA. In intestinal-type SBA, the mutation frequency of APC and CTNNB1 was 39%, closely aligning with the 45% aberrant expression of β-catenin. Aberrant expression of β-catenin and/or Wnt5a, a ligand of the noncanonical Wnt pathway, was detected in 60% of cases and showed a correlation with both cyclin D1 and c-Myc expression. Conclusions: These findings suggest that both canonical and noncanonical Wnt pathway-related proteins are involved in SBA carcinogenesis and progression. Notably, the canonical Wnt pathway appears to play a predominant role in intestinal-type SBA. Full article

Background/Objectives: Previous research has demonstrated that the initial letters of a word likely play a privileged role in visual word recognition, such that reading and visual recognition errors reflecting changes in this position are much less likely. For example, prior case studies of attentional dyslexia reported that participants were most accurate at rejecting nonwords formed by transposing a word’s first two letters (e.g., WONER from OWNER) compared to transpositions in later positions. The current study aimed to replicate and extend this finding in patients with posterior cortical atrophy (PCA), a neurodegenerative condition associated with visuospatial and attentional impairments. Methods: Two PCA patients completed lexical decision tasks involving five-letter real words and nonwords created either by transposing adjacent letters (in positions 1 + 2, 2 + 3, 3 + 4, or 4 + 5) or using matched nonword controls. To assess robustness, tasks were repeated across test–retest sessions. Stimuli were presented in both canonical horizontal and non-canonical vertical (marquee) formats. Accuracy, response bias, and sensitivity (d′) were estimated, with 95% confidence intervals derived from a nonparametric bootstrap procedure. Within-case logistic regressions were also conducted to illustrate the findings. Results: Both patients showed significantly higher accuracy and lower response bias for 1 + 2 transposition nonwords relative to other positions. This early-letter advantage persisted across test–retest observations and was maintained when words were presented in the vertical format, suggesting orientation-invariant effects. The bootstrap and regression analyses provided convergent support for these results. Conclusions: The findings provide novel evidence in PCA that the encoding of early letter positions operates independently of visual orientation and persists despite attentional deficits. This supports models in which the initial letters serve as a key anchor point in orthographic processing, highlighting the privileged and resilient status of early letter encoding in visual word recognition. Full article

Multiple myeloma (MM) is a complex genetic disease characterized by the heterogeneity of tumor cells. We have measured KRAS, NRAS, and BRAF gene mutations in circulating free tumor DNA (ctDNA) from plasma, bone marrow, and plasmacytoma samples as well as their correlation with various clinical and laboratory parameters. The prospective study included 113 MM patients (74 with plasmacytoma and 39 without), treated at the National Medical Research Center for Hematology (Moscow, Russia) from 2009 to 2024. FISH was performed on CD138+ bone marrow cells for 104 patients and array-CGH for two extramedullary plasmacytoma samples. Mutation analysis on CD138+ bone marrow cells was performed for 99 patients, on ctDNA for 80 patients, and, in 26 cases, samples of plasmacytoma were also investigated. Mutations in the KRAS, NRAS, and BRAF genes either in bone marrow, ctDNA, or plasmacytoma samples were found in 50% of patients. In patients with plasmacytoma, mutations in ctDNA were found in 28% of cases versus 0% in cases without plasmacytoma (p = 0.0007). Rare “noncanonical” KRAS and NRAS gene mutations were also more frequent in ctDNA compared to the bone marrow substrate (50% versus 9%, p = 0.01). Liquid biopsy in MM, particularly identification of the KRAS, NRAS, and BRAF gene mutations in ctDNA, is a valuable instrument for prognostication. Researching the intricate mechanisms underlying extramedullary involvement, and identifying novel high-risk factors associated with the disease, is worthwhile. Full article

Background and Clinical Significance: Patients with a neurodegenerative condition known as posterior cortical atrophy (PCA) can present with attention impairments across a variety of cognitive contexts, but the consequences of these are little explored in example of single word reading. Case Presentation: We present a detailed single-case study of KL, a local resident of South Wales, a patient diagnosed with posterior cortical atrophy (PCA) in 2018, whose reading and letter-naming abilities are selectively disrupted under non-canonical visual presentations. In particular, KL shows significantly impaired accuracy performance when reading words presented in tilted (rotated 90°) format. By contrast, his reading under conventional horizontal (canonical) presentation is nearly flawless. Whilst other presentation formats including, mixed-case text (e.g., TaBLe) and vertical (marquee) format led to only mild performance decrements—even though mixed-case formats are generally thought to increase attentional ‘crowding’ effects. Discussion: These findings indicate that impairments of word reading can emerge in PCA when visual-attentional demands are sufficiently high, and access to ‘top down’ orthographic information is severely attenuated. Next, we explored a cardinal feature of attentional dyslexia, namely the word–letter reading dissociation in which word reading is superior to letter-in-string naming. In KL, a similar dissociative pattern could be provoked by non-canonical formats. That is, conditions that similarly disrupted his word reading led to a pronounced disparity between word and letter-in-string naming performance. Moreover, different orientation formats revealed the availability (or otherwise) of distinct compensatory strategies. KL successfully relied on an oral (letter by letter) spelling strategy when reading vertically presented words or naming letters-in-strings, whereas he had no ability to engage compensatory mental rotation processes for tilted text. Thus, the observed impact of non-canonical presentations was moderated by the success or failure of alternative compensatory strategies. Conclusions: Importantly, our results suggest that an attentional ‘dyslexia-like’ profile can be unmasked in PCA under sufficiently taxing visual-attentional conditions. This approach may prove useful in clinical assessment, highlighting subtle reading impairments that conventional testing might overlook. Full article

Introduction: X-linked agammaglobulinemia (XLA) is a prototypical inborn error of immunity (IEI) caused by mutations in the BTK gene, leading to a profound deficiency of mature B cells and severe pan-hypogammaglobulinemia. The Epstein-Barr virus (EBV), which primarily infects B lymphocytes, is believed to be unable to establish persistence in these patients due to the lack of its natural reservoir. Indeed, current evidence supports that EBV infection is typically refractory in individuals with XLA. Methods: We describe the clinical and molecular characterization of a 10-year-old male patient with genetically confirmed XLA who developed EBV viremia, hemophagocytic lymphohistiocytosis (HLH), and EBV-positive cutaneous T cell lymphoma. Diagnosis was supported by flow cytometry, serology, quantitative PCR, EBER in situ hybridization, histopathology, and whole-exome sequencing. Results: Despite the complete absence of peripheral B cells, EBV was detected in leukocytes and multiple tissues, indicating active infection. The patient developed HLH and a T cell lymphoma with EBER-positive infiltrates. Genetic analysis revealed a nonsense mutation in BTK (1558C>T, R520*), confirming XLA. The clinical course included multiple episodes of neutropenia, viral and bacterial infections, and severe systemic inflammation. Conclusions: This is the first documented case of an XLA patient with confirmed BTK mutation presenting with clinical features more consistent with chronic active EBV infection. These findings challenge the prevailing paradigm that XLA confers protection against EBV-related diseases and further support the possibility of EBV noncanonical reservoirs leading to immune dysregulation. EBV should also be considered in the differential diagnosis of XLA patients presenting with systemic inflammation or lymphoproliferative disease. Full article