Search Results (773)

Ameloblastoma (AM) is a common locally invasive benign odontogenic tumor in Asian populations. Although fibroblast growth factor receptor 2 (FGFR2) mutations have been reported in AM, FGFR2 amplification, the predominant form of FGFR2 aberration in human cancers, remains unexplored. This study aimed to evaluate FGFR2 protein expression, FGFR2 gene copy number variations, and their associations with demographic and clinico-radio-pathological parameters in mandibular AM. Eighty-seven cases of mandibular AM and 10 dental follicle (DF) specimens were included. FGFR2 protein expression was assessed by immunohistochemistry, and gene copy number variations were analyzed using the quantitative real-time polymerase chain reaction (qPCR) technique. Clinical data, including age, gender, tumor size, radiographic features, histological subtype, and recurrence history, were examined for associations with FGFR2 alterations. FGFR2 protein overexpression was observed in 95.4% of AM cases and was not significantly associated with demographic or clinico-radio-pathological variables. FGFR2 gene amplification was detected in 52.5% of cases, while 8.2% showed gene deletion. Notably, 50.8% of cases exhibited concurrent FGFR2 amplification and overexpression, and all cases with FGFR2 gene deletion also demonstrated FGFR2 overexpression. These findings suggest that FGFR2 gene amplification and deletion may contribute to FGFR2 overexpression and play a significant role in the molecular pathogenesis of mandibular AM. Full article

The anticancer ruthenium complex indazolium trans-[tetrachlorobis(1H-indazole) ruthenate (III)—also known as KP1019—inhibits cancer cell proliferation in vitro, causes tumor regression in animal models, and showed no dose-limiting toxicity in a phase I clinical trial. Previous studies found that KP1019 damages DNA in both cancer cells and the budding yeast Saccharomyces cerevisiae. To identify other potential targets of KP1019 along with pathways that modulate the drug’s cellular effects, we screened the yeast gene deletion strain library by quantitative high-throughput cell array phenotyping (Q-HTCP). Fitness differences, as judged by growth curve analysis, identified genes for which loss of function (gene deletion) interacts with (enhances or suppresses) KP1019 effects. Drug-enhancing deletions were enriched for DNA repair functions, consistent with DNA damage being a primary target of KP1019 in yeast. pH homeostasis also modified the effects of KP1019. Drug-suppressing deletions prominently involved ribosomal proteins. A mechanistic link between ribosomal protein function and KP1019 toxicity was supported by dose-dependent accumulation of Rpl7a-GFP in the nucleolus, which is a hallmark of ribosomal biogenesis stress. Furthermore, KP1019 acted synergistically with the TOR pathway inhibitor everolimus to inhibit cell proliferation. The resulting model, wherein KP1019 perturbs ribosome assembly, can inform the design of future combination therapies. Full article

Background/Objectives: Canonical microRNAs possess a 5′ phosphate required for Argonaute binding and activity. However, prior work identified an unphosphorylated, inactive nuclear pool of the important radiation-responsive microRNA, miR-34, that is rapidly phosphorylated and activated in response to ionizing radiation (IR). Here, we extend this work and investigate the role of paraspeckles, a phase-separated nuclear sub-compartment, and their association with the localization of unphosphorylated miR-34a. Methods: Mass spectrometry was performed to identify interacting partners of unphosphorylated mir-34. CRISPR-mediated deletion of the paraspeckle NEAT1_2 triple helix motif was performed to create an A549 cell line lacking paraspeckles (dTH). Activity and expression of mir-34a post-irradiation were evaluated by qRT-PCR and luciferase assays comparing dTH and wild-type (WT) A549 cell lines. In situ hybridization (ISH) was performed to evaluate mir-34a localization before and after IR, comparing dTH and WT cell lines. Results: Mass spectrometry identified paraspeckle proteins as significantly enriched interacting partners of unphosphorylated mir-34 mimics. By qRT-PCR and luciferase assays, we found that paraspeckle loss prevented radiation-induced early activation of unphosphorylated mir-34a. We found no difference in radiation-induced transcription of pri-miR-34a, but early processing to pre-miR-34a appeared delayed. ISH confirmed that loss of paraspeckles altered the nuclear localization of miR-34a before and after IR. Conclusions: These data suggest that paraspeckles are associated with nuclear localization and early radiation-responsive activation of unphosphorylated miR-34a. This suggests a coordinated nuclear sequestration of this important miR in its unphosphorylated state to enable an enhanced radiation response. Full article

Boule is the ancestral member of the Deleted in Azoospermia (DAZ) family and is pivotal for gametogenesis and male fertility in most animals. However, there is a dearth of information on molluscan boule. Here, we identified a counterpart (Pcbol) from the genome of Pomacea canaliculata, which has emerged as a cosmopolitan alien species and notorious pest that causes devastating damage to aquatic biodiversity, freshwater ecosystems and crop production in invaded ranges. This study aimed to investigate the biological roles of Pcbol in male reproduction and to decipher the molecular mechanisms underpinning its modulation via dsRNA-delivered RNA interference (RNAi). The bioinformatic analysis showed that the Pcbol genomic sequence is 12,934 nt in length, harboring an open reading frame of 294 nt that encodes 97 aa residues, with an RRM domain evolutionarily conserved among molluscan orthologues. Spatiotemporal expression profiling indicated the predominant abundance of Pcbol in adult males and testis tissues. dsPcbol, injected at a dose of 4 μg/per snail for 5 days, yielded optimal silencing at both transcript and translation levels of Pcbol, as revealed by qRT-PCR and Western blotting. Immunofluorescence echoed a pronounced reduction in Pcbol signal intensity following RNAi. In addition to the arrested reproductive gland phenotype, the number of sperm cells substantially dwindled upon dsPcbol treatment relative to the dsGFP control. In biochemical and fecundity assays, Pcbol depletion triggered a significant decrease in Te/SP/Arg content and suppressed the number of deposited eggs and hatchability. Furthermore, spermatogenic genes like CDC25/TSSK1/SPATA17/DDX4/Dmrt2/Sox2/Kelch10/SPO11 displayed considerable downregulation post Pcbol silencing, with molecular docking predicting a strong affinity between CDC25 and Pcbol. These molecular modules may interact with Pcbol to mediate knockdown effects on spermatogenesis dysfunction. Collectively, our findings not only confirmed that boule was indispensable for spermatogenesis and male fertility in a mollusk, but also highlighted the Pcbol-based male sterile technique (MST), which can be incorporated into precision pest management (PPM) strategies for sustainable control of P. canaliculata. Full article

Listeria monocytogenes (Lm) is a foodborne pathogen whose virulence depends on the coordinated action of multiple virulence factors. Although deletion of either LIPI-4 or inlB reduces the virulence of Listeria monocytogenes, it remains unknown whether these two factors are functionally or regulatory connected. Therefore, we constructed an inlB deletion mutant and its complemented strain in the Lm928 and ΔLIPI-4 backgrounds. We assessed bacterial growth, biofilm formation, motility, host cell interactions (adhesion, invasion, intracellular proliferation), plaque formation, mouse organ colonization. Growth curve analysis showed no significant differences among strains. qPCR revealed that LIPI-4 modulates inlB expression in a cell-type-specific manner: inlB was downregulated in ΔLIPI-4 under culture and HTR-8 infection, but upregulated during hCMEC/D3 infection—yet functional defects persisted in all cases. Biofilm assays showed that ΔLIPI-4 and the double mutant exhibited enhanced biofilm formation, with the double mutant exceeding ΔLIPI-4, demonstrating synergistic enhancement. Motility assays indicated that LIPI-4 dominates bacterial movement, with ΔLIPI-4 and the double mutant showing identical severe defects. Plaque formation analysis showed that LIPI-4 is essential for cell-to-cell spread, while inlB deletion unexpectedly enhanced plaque formation—an effect completely abolished in the absence of LIPI-4. Host cell assays across Caco-2, HTR-8, and hCMEC/D3 models revealed that LIPI-4 is the core determinant of adhesion, invasion, and intracellular proliferation, whereas inlB contributes in the context of LIPI-4 and its effects vary with the specific cellular process examined. In mice, LIPI-4 was essential for systemic colonization of the liver and spleen, with inlB acting as a co-factor, whereas inlB unexpectedly promoted higher bacterial burdens in the brain, suggesting that inlB modulates LIPI-4-mediated neuroinvasion. Overall, our results establish LIPI-4 as the central determinant of Lm virulence, with inlB acting as a context-dependent co-factor that modulates LIPI-4-mediated pathogenesis in a cell type- and tissue-specific manner. Full article

H-NS (histone-like nucleoid-structuring protein) is a global regulator affecting diverse bacterial processes. This study aimed to elucidate the regulatory role of H-NS in the virulence of Klebsiella pneumoniae (K. pneumoniae), particularly in relation to capsule synthesis and anchoring. A clinically isolated ST11-KL64 strain of K. pneumoniae FK6741 with low virulence was used. The role of H-NS was evaluated using colony morphology, the string test, viscosity measurement, capsule quantification, transmission electron microscopy, growth curve, biofilm assay, a mouse infection model, transcriptomic analysis, and RT-qPCR. Deletion of hns converted FK6741 into a hypermucoid phenotype in the positive string test; capsule quantification and transmission electron microscopy (TEM) showed increased polysaccharide chains but a reduced and tightly bound capsule. The mutant was initially found to grow slowly but formed stronger biofilms. In vivo, it displayed reduced virulence but induced stronger inflammation. Molecular assays revealed upregulation of capsule synthesis genes (galF, wzi, wcaJ, and wzc) and downregulation of wabG, which is involved in capsule anchoring. H-NS represses capsule synthesis genes, limiting capsule formation in K. pneumoniae. In contrast, loss of H-NS downregulates wabG, a key gene involved in GalA-mediated capsule anchoring, resulting in unstable surface attachment and loss of capsular polysaccharides. Consequently, these unanchored polysaccharides fail to confer effective protection, resulting in reduced bacterial virulence. Full article

Vibrio anguillarum is a major pathogenic bacterium causing vibriosis in aquatic animals, leading to substantial economic losses in the global aquaculture industry. Previous studies have indicated that L-arginine modulates the virulence of the pathogen, but the underlying molecular mechanisms remain elusive. The present study aimed to clarify the regulatory role of L-arginine metabolism in V. anguillarum virulence. We first evaluated the effects of L-arginine and its major metabolites (agmatine, putrescine, spermine) on the hemolytic activity of V. anguillarum. Results showed that L-arginine and its metabolites regulated hemolytic activity in a concentration-dependent biphasic manner, with agmatine exerting the most potent promoting effect. To identify the critical metabolic branch involved, four isogenic mutants were constructed targeting key genes in arginine metabolism (adc, astA, astD). Phenotypic analysis revealed that only the adc deletion mutant (Δadc) exhibited near-complete loss of hemolytic activity, which was dose-dependently restored by supplementation with agmatine, putrescine, or spermine. Transcriptomic analysis identified 704 significantly differentially expressed genes (DEGs) between Δadc and WT strains, with downregulated DEGs enriched in virulence-associated pathways. Key hemolysin and secretion system genes were validated to be downregulated in ∆adc by quantitative real-time PCR (qRT-PCR). Additionally, Δadc displayed attenuated anti-phagocytic ability in Tetrahymena co-culture assays, impaired biofilm formation, and increased susceptibility to multiple classes of antibiotics. Collectively, our findings demonstrate that L-arginine modulates V. anguillarum hemolysis and overall virulence through the adc-mediated agmatine biosynthesis branch. This study fills the knowledge gap in the regulatory mechanism of L-arginine on V. anguillarum virulence and provides a potential target for the control of vibriosis in aquaculture. Full article

Apple replant disease (ARD) is a soil-borne disease that severely restricts root development in orchards, impedes tree growth, and leads to reduced yields and decreased fruit quality, and thus significant economic losses. Previous studies identified Fusarium oxysporum as a major pathogenic agent. In this study, a T-DNA insertion mutant library of 13,000 F. oxysporum HS2 strains was utilized to screen for mutants with impaired pathogenicity. Nine mutants exhibiting reduced virulence were obtained, and the insertion sites of five mutants were successfully identified. Among them, we selected the HS2-29 strain, which exhibited the most significant decrease in conidial production, for further investigation. Its T-DNA was inserted into the FoPLT gene. RT-qPCR analysis revealed that the expression of the FoPLT gene rapidly increased during the early infection stage, followed by a decline and eventual stabilization. After the deletion of the FoPLT gene, the production of aerial hyphae, conidial yield, conidial length, and conidial diameter all significantly decreased. Stress tolerance assays indicated that FoPLT does not affect cell wall integrity in F. oxysporum. The deletion of the FoPLT gene significantly reduced the pathogenicity of F. oxysporum, and inoculating Malus robusta seedlings with the FoPLT knockout mutant led to significant increases in plant height, root length, fresh weight, and dry weight. These results suggest that the FoPLT gene plays a critical role in the pathogenicity of F. oxysporum. Full article

Background: 22q11.2 deletion syndrome (22q11.2 DS) is a rare genetic syndrome characterized by high phenotypic variability, with an incidence of approximately 1:4000 live births. Most of the existing literature consists of case reports or case series, making it challenging to obtain large cohorts for data comparison and drawing generalizable conclusions. Aim: The aim of this article is to share the clinical experience of patients with 22q11.2 DS who were previously evaluated by Child Neuropsychiatry and underwent odontoiatric procedures at the Dental Unit of the Policlinico di Bari. Methods: We report three clinical cases of pediatric patients with 22q11.2 DS who were managed at the University Dental Unit of the Policlinico di Bari for dental procedures, including pre/post-treatment images and child neuropsychiatric characteristics. Results: Cleft lip and cleft palate are the most commonly encountered facial malformations. Enamel hypoplasia and hypomineralizations, caries, dental agenesis, and hypodontia are reported with variable frequency in almost all studies considering the oral health of these patients. Our experience is coherent with the data present at the moment. The clinical examinations showed diffuse hypomineralization and several caries, concordant with the literature. One patient received moderate sedation and another received general anesthesia; oral prophylaxis, fluoride application, pulp therapy, extractions of hopeless teeth and restorations of carious and hypomineralized teeth were performed. Conclusions: 22q11.2 DS is a genetic condition with a variable prognosis, but current trends show that patients often reach adulthood, primarily due to advancements in cardiology, which was previously the leading cause of mortality in these patients. From this perspective, collaboration among various medical specialties is crucial to implement holistic patient management programs and early interventions. Full article

Background: Sirtuin 1 (Sirt1) is known to regulate stem cell differentiation and cardiomyocyte function, yet its specific role and mechanism in human embryonic stem cell (hESC) differentiation into cardiomyocytes remain unclear. This study aimed to elucidate the functional contribution and molecular pathway of Sirt1 in cardiomyogenesis. Methods: A Sirt1 knockout (Sirt1⁻/⁻) hESC line was generated using CRISPR-Cas9 technology. The expression of key differentiation markers was analyzed by RT-qPCR at days 6, 8, and 9. The underlying mechanism was investigated through integrated RNA-sequencing (RNA-seq) analysis and dual-luciferase reporter assays. Results: Sirt1 deletion significantly downregulated the expression of mesodermal (TBX6, KDR), cardiac precursor (NKX2.5, TBX5), and mature cardiomyocyte (cTNT, Hand2) markers. Mechanistically, a competing endogenous RNA (ceRNA) axis, LncRNA XR_951230.1/miR-3663-3p/SMYD1, was identified. Sirt1 knockout reduced XR_951230.1 expression, which consequently elevated miR-3663-3p activity and suppressed its target gene SMYD1. Conclusions: These findings indicate that Sirt1 is essential for promoting hESC differentiation into cardiomyocytes, potentially via the XR_951230.1/miR-3663-3p/SMYD1 pathway. This study provides new insights into the regulatory network of stem cell-based cardiomyogenesis and suggests potential targets for stem cell-based cardiac disease therapy. Full article

Objective: Copy number variants (CNVs) overlapping genes associated with neurodevelopmental disorders in patients with epilepsy are particularly concentrated in epilepsy hotspot loci. The aim of this study was to evaluate epilepsy as a component of the dysmorphic–neurodevelopmental phenotype in patients with recurrent CNVs. Methods: The study included genetic and clinical data from 177 pediatric patients carrying 17 recurrent CNVs showing well-documented enrichment in epilepsy or associated with genetic OMIM syndromes. Results: Epilepsy was diagnosed in 50 of 177 children (28.2%), developmental delay in 147 (83.0%), dysmorphic features in 104 (58.8%), behavioral problems in 62 (35.0%), and congenital anomalies in 55 (31.1%). Among recurrent CNV hotspots, the del16p11.2 BP4–BP5 deletion was the most frequent, occurring in 39 of 177 patients. Ten children (25.6%) with del16p11.2 presented with epilepsy as part of the phenotype. Other frequently represented CNVs included del15q11.2 BP1–BP2 (OMIM #615656; 19/177 patients, 4/19 with epilepsy), del1q21.1 (OMIM #612474; 15/177, 6/15 with epilepsy), del15q13.3 (OMIM #612001; 13/177, 4/13 with epilepsy), and dup16p11.2 (OMIM #614671; 12/177, 1/12 with epilepsy). The highest proportion of epilepsy as a phenotypic component was observed in patients with del1p36 (OMIM #607872; 6/9 patients) and del1q21.1 (OMIM #612474; 6/15 patients). Conclusions: Our data support the clinical utility of CNV testing in patients with epilepsy accompanied by additional neurodevelopmental or dysmorphic features, in line with current diagnostic guidelines. The epilepsy-plus phenotype may help clinicians identify patients who are most likely to benefit from CNV analysis. Full article

Background: 22q11.2 deletion syndrome (DiGeorge Syndrome) is a rare disorder that involves a de novo hemizygous microdeletion within the 22q11.2 chromosomal locus. Individuals affected by this condition display a wide array of clinical phenotypes as well as haplotype sequences, which render understanding the genotype–phenotype relationship quite difficult. Additionally, the complex structure of the 22q11.2 low-copy repeats (LCRs), which usually inhibits sequencing efforts, has complicated the study of possible breakpoints that instigate the deletion events. In this study, 22q11.2 deletion syndrome is investigated on a genomic and phenotypic level for the purpose of determining the impact of each deletion type and identifying possible candidate breakpoints. Methods: In the present study, a systematic review combined with a secondary genomic analysis has been executed following PRISMA guidelines using PubMed and Scopus publications in order to estimate its holistic genomic map, genomic functional elements, and key genomic regions such as LCRs. A statistical content analysis of the affected chromosomal regions was also performed. Groups of functional elements with common traits were composed, and their contribution to the deletion events was investigated. Finally, the 22q11.2 repeat regions were screened for palindromic AT-rich repeats. Results: Of the 8202 unique publications studied in this work, only 65 met the inclusion criteria. The estimated genomic map of 22q11.2 deletion syndrome in the secondary genomic analysis revealed 11 distinct microdeletions occurring between eight LCRs, and a new repeat region within the CES region (CESRR), of which the LCR22A-LCR22D deletion was the most frequently reported. Last but not least, the palindromic analyses indicated eight critical groups as candidate breakpoints that potentially form four distinct patterns, and ten palindromic AT-rich repeat (PATRR) regions were identified amongst LCR22A, LCR22B, LCR22D, LCR22F and LCR22H. Conclusions: The study results validate the differentiating clinical contribution between the proximal and the distal segments. Eight novel candidate breakpoints and five new PATRRs were identified that require further study to establish their involvement in 22q11.2 microdeletion events. Full article

Despite increasing reports of zoonotic simian foamy virus (SFV) infections globally, knowledge of its genetic adaptation in humans and impact on viral transmission and pathogenicity remains limited. We obtained complete SFV genomes using metagenomics analysis of viral isolates from peripheral blood lymphocytes (PBLs) and throat specimens from a worker (Case 6) and source chimpanzee (B1) that bit him. We analyzed viral diversity in three genomic regions (LTR, tas, and bet) involved in replication and latency using longitudinal specimens (PBLs, throat, saliva, urine, and semen) from Case 6 over five years, and PBLs from B1 and five additional chimpanzees over three years. Proviral loads were measured using a validated qPCR assay. Phylogenetic analysis revealed nearly identical SFV genomes in Case 6 and B1. Overall, bet sequences exhibited high genetic stability across body compartments and over time, with evidence of compartmentalization in Case 6 urine and semen specimens. G→A substitutions in GG and GA motifs in bet indicated heterogeneous APOBEC-associated editing across hosts and anatomical compartments following zoonotic transmission. Case 6 had significant deletions in the LTR region that were absent in B1 and other chimpanzees. Length variation in tas, including truncated forms, was observed across longitudinal specimens from Case 6, B1, and other chimpanzees. Proviral loads were consistently low and undetectable in most Case 6 urine specimens. Together, analysis of this SFV transmission pair identifies genomic changes likely to affect viral replication and persistence, highlighting mechanisms that may limit secondary transmission and pathogenicity of SFV in humans. Full article

Background: Chromosome 3p (chr3p) is frequently deleted in multiple cancers, indicating the presence of shared tumor suppressors. In aggressive uveal melanomas (UVM), this deletion often co-occurs with chr8q amplification (8q+), suggesting strong selection pressure during UVM evolution. Methods: To understand the pattern of genomic alterations mediated by chr3p deletion, we have developed an algorithm for detecting isochromosomes in 10,632 TCGA cancer patients. We further perform integrative genomics analysis to investigate how chr3p deletion could affect subsequent cancer genome evolution and synthetic lethality in UVM. Results: Analysis of genomic alterations in 33 different cancer types implicates the deletion or deleterious mutations of SET-domain-containing 2 (SETD2) at chr3p21 in significantly facilitating the formation of isochromosomes, thereby promoting genomic instability conducive to rapid cancer genome evolution. Fracturing of dicentric isochromosomes during cell division is pervasive and follows the dynamic fragmentation pattern of solids under impulse. In the most aggressive UVM subtype, chr3 deletion includes MITF, a master regulator of melanocyte survival and differentiation, and co-occurs with 8q+. We demonstrate that MITF is a master transcriptional regulator of GNAQ/GNA11 and associated synthetic-lethal genes in UVM. MITF maintains MAPK and calcium homeostasis in UVM, and its hemizygous deletion is thus accidental, likely creating an early crisis during oncogenesis. We further show that MITF, MYC, and GNAQ/GNA11 form coupled regulatory feedback loops in the melanocyte lineage, and MITF deletion in UVM creates acute dependency on MYC-mediated rescue via 8q+. The discovered feedback loops predict both overall and relapse-free patient survival within the most aggressive UVM subtype, explain sensitivity to therapeutic gene perturbations, and inform effective combinatorial therapies. Conclusions: SETD2 deletion potentiates isochromosome formation across diverse cancers. Combinatorial targeting of MITF together with a previously identified synthetic lethal gene may benefit UVM patients harboring both chr3 deletion and 8q+. Full article

Aspergillus flavus Link, 1809 is a pathogenic fungus widely present in the environment. It can infect plants and also acts as an opportunistic pathogen affecting humans and other animals. The aflatoxins (AFs), it produces, can cause cancers such as liver cancer. Therefore, in-depth research into the pathogenic mechanisms of A. flavus is crucial. Arp8 (Actin-like protein Arp8) is a unique subunit within the chromatin remodeling complex INO80, regulating processes including chromatin remodeling. However, the biological function of Arp8 in A. flavus remains unclear. This study constructed A. flavus arp8 knockout (Δarp8) and complementation (Com-arp8) strains via homologous recombination. Subsequent research revealed that following the deletion of arp8, A. flavus exhibits a reduction of approximately 51% in conidia production, complete abrogation of sclerotia formation, and significantly impairment of aflatoxin B1 (AFB1) biosynthesis. Crop grain colonization and Bombyx mori Linnaeus, 1758 infection models demonstrated that Arp8 plays a crucial role in A. flavus ability to infect hosts. Environmental stress experiments identified Arp8 as a vital factor for A. flavus in response to various environmental stresses. Quantitative RT-PCR (qRT-PCR) analysis indicated Arp8 achieves its biological functions through corresponding regulatory factors. This study elucidates the biological functions of Arp8 in A. flavus growth and development, pathogenicity, and aflatoxin synthesis, laying a foundation to illuminate the mechanisms of A. flavus pathogenicity and AFs production. Full article