Antibiotic exposure promotes the emergence of antimicrobial resistance by activating SOS-dependent mutagenesis and facilitating horizontal gene transfer (HGT), both of which accelerate bacterial adaptation under selective pressure. Consequently, pharmacological inhibition of the SOS response has emerged as a promising strategy to suppress resistance evolution and enhance the efficacy of existing antibiotics. In particular, proteins involved in homologous recombination and DNA repair, such as RecA and LexA, represent attractive therapeutic targets for preventing SOS activation and limiting mutagenesis. This review highlights the pivotal role of the bacterial SOS response in antimicrobial resistance and discusses emerging strategies to therapeutically target the SOS pathway for combating resistant pathogens.
Background/Objectives: Non-geniculate coralline red algae of the order Sporolithales are ecologically important calcium carbonate producers in tropical and subtropical seas globally; however, molecular surveys of this group in the Bay of Bengal (BoB) are entirely lacking, and no species of Sporolithon Heydrich has been documented from this embayment. Here we report the first occurrence of Sporolithon from Saint Martin Island, the sole coral island of Bangladesh, at the northernmost margin of the BoB. Methods: Seven specimens encompassing free-living rhodoliths to encrusting growth forms were collected from intertidal and shallow subtidal habitats in March 2024. Species identification was assessed by combining scanning electron microscopy (SEM) images with a six-locus molecular dataset comprising three plastid-encoded markers (rbcL, psbA, and UPA), the mitochondrial cytochrome c oxidase subunit I 5′ region (COI-5P), and two nuclear-encoded ribosomal markers (LSU and SSU). Results: Both maximum likelihood and Bayesian phylogenetic analyses resolved all BoB specimens within a well-supported clade of Sporolithon indopacificum Maneveldt, Gabrielson & Kangwe, with 0.00–0.20% concatenated rbcL + psbA sequence divergence from type specimens from Tanzania and previously published sequences from Fiji, China, and Guam (USA). SEM confirmed morpho-anatomical features concordant with the original species description, including a free-living rhodolith habit, monomerous construction, and tetrasporangial compartments with apical pore plugs. Conclusions: This record extends the confirmed range of S. indopacificum into the northeastern Indian Ocean, represents the first documented Sporolithon occurrence in Bangladeshi waters, and highlights the value of combining SEM with multi-locus barcoding for coralline algal surveys in undersampled tropical regions.
Background: Oral leukoplakia is a clinically important oral potentially malignant disorder with variable risk of progression to oral squamous cell carcinoma. Histopathological grading of oral epithelial dysplasia remains the primary method for risk assessment; however, its predictive accuracy is limited by sampling variability, lesion heterogeneity, and interobserver variation. DNA aneuploidy and abnormal DNA ploidy status have therefore been investigated as objective biomarkers of genomic instability and potential predictors of malignant transformation. Aim: This narrative review evaluates whether DNA aneuploidy or abnormal DNA ploidy status predicts malignant transformation of oral leukoplakia to oral squamous cell carcinoma compared with diploid DNA status, and whether DNA ploidy should be interpreted as a stand-alone prognostic marker or as part of combined risk-assessment models. Materials and Methods: A structured PubMed/MEDLINE search (last performed on 12 July 2026) identified 162 records using terms related to oral leukoplakia, oral potentially malignant disorders, DNA ploidy, DNA aneuploidy, cytometry, malignant transformation, and oral squamous cell carcinoma. After screening and full-text eligibility assessment, 45 studies were included and organised according to their evidential role as core prognostic studies, PVL-specific subgroup evidence, and supporting/background evidence addressing diagnostic relevance, dysplasia correlation, treatment monitoring, genomic instability, and methodological development. Results: Most core prognostic studies showed that DNA aneuploidy, abnormal DNA content, or chromosomal instability was associated with increased malignant transformation risk compared with diploid or non-aneuploid status. Several studies reported higher transformation rates, hazard ratios, or improved prediction when DNA ploidy was combined with dysplasia grading, lesion site, clinical heterogeneity, or other biomarkers. However, predictive performance varied across studies, and DNA ploidy alone often showed modest sensitivity, specificity, or positive predictive value. Diploid or non-aneuploid status appeared more useful for identifying lower-risk lesions, although it did not completely exclude malignant transformation. PVL evidence suggested frequent aneuploidy and aggressive genomic behaviour, but DNA ploidy appeared less reliable for discriminating risk within PVL. Conclusions: DNA aneuploidy and abnormal DNA ploidy status are meaningful markers of genomic instability and are associated with increased malignant transformation risk in oral leukoplakia. However, when used alone, DNA ploidy often shows only modest predictive performance, and prediction improves consistently when it is combined with dysplasia grading and other clinicopathological factors. Their strongest clinical value is therefore as adjunctive biomarkers within combined clinicopathological and molecular risk models, rather than as stand-alone replacements for histopathological assessment; routine clinical implementation is not yet supported by the current evidence.
Background/Objectives: Oxford Nanopore sequencing produces long reads quickly, but most functional profiling tools were developed for short reads or rely on assembly pipelines that are computationally costly and sensitive to long-read error rates. We present NanoPrism, a taxonomy-guided pipeline for rapid functional profiling of long-read metagenomes. Methods: NanoPrism (i) identifies sample composition with Kraken2, (ii) constructs compact species-specific coding sequence (CDS)–KEGG ortholog databases, and (iii) estimates ortholog abundances by direct minimap2 alignment of nanopore reads with single-copy marker normalization. We evaluated NanoPrism on simulated Pseudomonas aeruginosa PAO1 and PA14 reads and on ZymoBIOMICS mock-community datasets sequenced on GridION and PromethION platforms. Results: On the Zymo long-read datasets, NanoPrism achieved Pearson correlations of 0.917–0.922 against independent expected ortholog profiles under unit-sum normalization. On matched one-million-read subsets, NanoPrism achieved higher correlations and lower Jensen–Shannon distances and mean absolute errors than the evaluated DIAMOND-based MEGAN-LR workflow. Experiments that omitted one species at a time from the reference database showed that omission of low-abundance community members had limited effects on the aggregate KO profile, whereas omission of the dominant Listeria monocytogenes reference from the Log community reduced Pearson correlation from approximately 0.92 to 0.29. Conclusions: NanoPrism offers a computationally efficient option for taxonomy-guided functional profiling of bacterial isolates and defined microbial communities. Validation on complex clinical and environmental metagenomes, broader forms of taxonomic-classification error, and dedicated fungal benchmarks remain necessary.
Eosinophilic esophagitis (EoE) and inflammatory bowel disease (IBD) are immune-mediated disorders of the gastrointestinal (GI) tract that, despite involving different tissues, are increasingly recognized to coexist. Epidemiologic studies demonstrate a bidirectional association, with patients affected by one condition showing a higher-than-expected prevalence of the other, suggesting shared susceptibility rather than incidental overlap. Genetic and epigenetic data support partial convergence in immune regulatory pathways, while epithelial barrier dysfunction and antigen-driven immune activation emerge as common upstream features. Overlapping cytokine networks, including IL-4, IL-13, and IL-23 signaling, contribute to chronic inflammation in both diseases, although differences in tissue environment and immune dominance give rise to distinct inflammatory phenotypes and clinical behavior. Clinical outcomes in patients with dual diagnoses appear heterogeneous, with available data suggesting neither uniformly worsened nor clearly protective disease courses, underscoring the complexity of shared immune mechanisms operating within different anatomic contexts. Beyond inflammatory activity, coexistence of EoE and IBD poses important nutritional and quality-of-life challenges, as overlapping dietary restrictions and chronic symptoms increase the risk of malnutrition, micronutrient deficiencies, and psychosocial burden. Current therapies remain disease-specific, with strong evidence supporting proton pump inhibitors, swallowed topical steroids, dietary therapy, and dupilumab in EoE, and biologics and small molecules targeting TNF-α, IL-12/23, IL-23, integrins, and JAK–STAT signaling in IBD, while evidence guiding treatment in patients with dual diagnosis remains limited. Together, current evidence supports a framework of shared immune machinery with tissue-specific expression that explains coexistence while preserving the distinct identities of EoE and IBD. By integrating emerging genetic, immunologic, and clinical evidence, this review aims to provide a framework for understanding and managing patients with coexisting EoE and IBD.
Real-time selective sequencing on nanopore platforms offers a programmable way to enrich target molecules and deplete background DNA during a run. This approach, widely known as adaptive sampling (AS), has been applied across host depletion, metagenomics, targeted loci, plasmid/AMR workflows, and RNA/transcriptomic protocols, but reported performance varies substantially across studies. This review synthesizes current algorithmic and empirical evidence with emphasis on sequencing-relevant outcomes, including absolute informative yield, target-coverage behavior, throughput effects, and run-to-run stability. Across use cases, relative enrichment is frequently observed, but gains in usable genomic output are strongly conditioned by fragment-length distributions, reference quality, decision-loop latency, and rejection-associated penalties in total yield and pore longevity. Evidence from targeted-panel and complex-locus studies further indicates that improved depth concentration can coexist with coverage non-uniformity and context-specific trade-offs relative to wet-lab enrichment. Overall, the literature supports AS as a valuable but condition-dependent strategy whose benefit is greatest when assay design, reference selection, and computational constraints are jointly optimized.
Epigenetics is a widely present mechanism for the modulation of gene expression without alterations in the underlying genetic sequence. Epigenetic signatures are significantly present in bacteria, with DNA methylation playing a key role in the modulation of bacterial physiology and pathogenesis. DNA methyltransferases (MTases) are the enzymes catalyzing the transfer of methyl groups to adenine or cytosine residues in the DNA using the methyl donor S-adenosyl-L-methionine (SAM). This process generates modified bases, N6-methyladenine (m6A), 5-methylcytosine (5mC), or N4-methyl cytosine (4mC) in the DNA, which influence fundamental cellular processes such as DNA transactions, DNA replication, transcription, and DNA repair. These MTases, earlier thought to be a part of primitive bacterial immune system, are now considered to be active players in gene regulation. They regulate bacterial adaptability to stress by virtue of phase variation and bistability. In pathogenic species such as Mycobacterium tuberculosis (Mtb), DNA methylation driven epigenetic reprogramming influences the expression of virulence factors, antibiotic tolerance, and persistence genes. This review gives a detailed account of role of DNA methyltransferases in bacterial epigenomics influencing various cellular processes. With the development of long-read high-throughput sequencing technologies, single-base mapping of bacterial methylomes has become possible. In the latter part of the review, we talk about these advances and the integration of synthetic biology to expand the potential of methylation systems for developing biosensors and switchable gene expression platforms. These strategies can be translated into future vaccine design and precision drugs for disease control. Deciphering bacterial DNA methylation can help gain insights into microbial evolution and design innovative therapeutics for various diseases.
Background: Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression, playing pivotal roles in diverse biological processes, including reproduction. This study identified and characterized lncRNAs located near fertility-associated genes in Retinta beef cattle, exploring their potential regulatory roles via DNA-RNA triplex formation using in silico approaches. Methods: We applied an integrative bioinformatics pipeline to identify potential triplex interactions, predicting structurally accessible regions within the lncRNAs and demonstrating the statistical enrichment of binding sites across known regulatory genomic elements. Results: Twelve protein-coding genes previously linked to female fertility or male scrotal circumference were analyzed, revealing 16 unique lncRNAs within +/- 50 kb windows, predominantly on BTA5. We predicted high-confidence triplex-forming oligonucleotides (TFOs) for most gene-lncRNA pairs. Our results suggest robustness and sequence specificity, as interactions were disrupted by sequence permutation or when a control background sequence was used. RNA secondary-structure analysis revealed that TFOs generally lie in exposed regions, supporting their accessibility for triplex formation. Furthermore, promoter and regulatory regions of fertility-associated genes were enriched in predicted triplex target sites (TTSs), with some overlapping CpG islands and enhancer regions, leading to the hypothesis that these lncRNAs might play a role in epigenetic regulation. Conclusions: Overall, these findings establish computationally derived hypotheses regarding the potential molecular mechanisms by which lncRNAs may modulate reproductive efficiency in cattle and highlight specific lncRNAs as promising targets for functional studies and marker-assisted breeding.
Introduction: Does DNA superhelicity effect nucleic acid interactions with drugs? To test such a possibility, the interactions of the linear and superhelical forms of the pGEX-4T-2 plasmid have been investigated with a newly synthesized compound, 4-chloro-6-(1H-imidazo [4,5-b]phenazine-2-yl)benzene-1,3-diol, positively tested for the antiproliferative (cell growth-limiting) properties, important for the development of anticancer drugs. Methods: The accumulation of the compound, and its possible reorientation (phase transition) within the plasmid layer adsorbed on a Glassy Carbon (GC) electrode has been monitored in 5 min. intervals using alternatively two voltammetric methods-Differential Pulse (DP), showing redox properties and-Alternating Current (AC), reflecting both redox and structural properties (capacity/resistance change related to DNA condensation) of the DNA-drug layers. Results and Discussion: The accumulation plots of the compound in plasmid layers are different for superhelical and linear pGEX-4T-2 and also depend on the DNA coverage by the compound. The reorientation (phase transition) occurs at a compound concentration 2 & micro;M for the superhelical plasmid and 1.5 & micro;M for the linear one, as compared to 8 & micro;M for the compound layer formed on bare GC, thus proving the existence of the DNA-compound interactions. Interestingly, the phase transition is redox-sensitive, e.g., AC redox signal II is visible for the linear, but not for the superhelical plasmid, thus reflecting different orientation of the compound in these two types of pGEX-4T-2 plasmid, related to the condensation in the DNA-compound layer and seen as a decrease in the C/R signal. Conclusions: The results suggest that the reorientation of the compound leading to plasmid condensation occurs differently in supercoiled and linear pGEX-4T-2 (redox specific accumulation and condensation).
Lodgepole pine (Pinus contorta Dougl.) exhibits pronounced morphological variation across its range, historically attributed to allopatric differentiation during the Wisconsin glaciation. However, whether genetic divergence aligns with morphological differentiation-a fundamental prediction of allopatric speciation theory-remains untested. We conducted a comprehensive phylogeographic analysis of chloroplast DNA (trnL intron and trnL/trnF spacer) and mitochondrial DNA (nad1 b/c intron) across 31 populations representing all four recognized subspecies to test hypotheses of refugial isolation and to evaluate the genetic basis of current taxonomic classification. Contrary to predictions of allopatric divergence, both organellar genomes showed striking genetic uniformity (pi = 0.000178-0.000186; intersubspecific genetic distances: 1.06 & times; 10(-4) to 3.96 & times; 10(-4)) with no phylogenetic structure corresponding to morphological boundaries. Significant negative neutrality test values (Tajima's D = -2.26, p < 0.02; Fu and Li's D* = -4.52, p < 0.02) suggest recent demographic expansion rather than equilibrium divergence. A distinctive 5 bp indel in coastal populations provides molecular evidence for a northern Pacific refugium, and its occurrence in interior populations is consistent with post-glacial pollen-mediated gene flow, though this directionality remains inferential pending nuclear genomic confirmation. These findings suggest that morphological divergence reflects rapid adaptive evolution in heterogeneous environments rather than deep phylogenetic divisions. This pattern exemplifies gene flow-selection balance, in which divergent selection maintains local adaptation despite extensive gene flow-supporting an ecotypic rather than a phylogenetic interpretation of intraspecific diversity. The persistence of morphological variation despite genetic homogeneity indicates strong selection on ecologically important traits, likely driven by variation in fire regimes, differential seed predation, and climate gradients. These results have critical implications for understanding adaptive evolution rates in widespread conifers and for developing conservation strategies that emphasize adaptive processes over taxonomic categories.
Cancer cells have many derailed processes due to which they have a higher proliferative capacity. The rewiring is continuously taking place to meet their metabolic demands. The demands depend on the stage of cancer, and these differences create challenges in curing them. Nucleotide metabolism plays a pivotal role in shaping cancer fate. DNA repair and other damage pathways also play a key role in cancer progression, genomic instability, errors in genetic material etc. These are discussed in this mini review so that researchers can take the lead to make an effort to combat cancer and design new therapeutics.
Gut dysbiosis, defined as a disruption in the structure or function of the intestinal microbiota, is increasingly recognized as a key contributor to inflammatory, metabolic, and neuropsychiatric diseases. Conventional interventions such as broad-spectrum antibiotics, generic probiotics, and fecal microbiota transplantation (FMT) often show limited and inconsistent efficacy because they lack specificity, durability, and robust safety controls. In contrast, recent advances in DNA-based technologies are reshaping the therapeutic landscape by enabling targeted, programmable, and mechanistically informed modulation of the gut ecosystem. This review presents an integrated overview of three major domains driving this shift: CRISPR-based systems that selectively delete, silence, or reprogram microbial genes; synthetic biology-driven live therapeutics engineered to sense disease-associated cues and execute controlled responses; and metagenomics-informed strategies that tailor interventions to patient-specific microbial gene profiles and functional deficits. Additionally, we examine the continued evolution of FMT toward DNA-optimized workflows and defined microbial consortia that offer safer, more standardized alternatives to crude donor material. Across these domains, we discuss delivery platforms (including bacteriophages, conjugative plasmids, extracellular vesicles, and synthetic nanoparticles), and compare their efficiency, specificity, and scalability. We further highlight how DNA-guided interventions interface with host immunity—shaping Treg/Th17 balance, mucosal barrier function, and inflammatory signaling—while also analyzing ecological and evolutionary risks, biocontainment strategies, and regulatory classification gaps that will govern clinical translation. Together, these developments signal a transition from empirical microbiome manipulation to rational ecosystem engineering. DNA-guided therapies hold strong promise for precise and personalized management of gut-related diseases, but their success will depend on rigorous ecological risk assessment, long-term monitoring, and adaptive regulatory frameworks alongside continued technological innovation.
Background: Piper is one of the largest genera in the family Piperaceae, with approximately 2100 species. Most Piper species are used as spices or as medicinal plants. Piper methysticum G. Forst., popularly known as kava-kava (or kava), is widely used to treat anxiety disorders. Due to similar morphological features, P. auritum Kunth (known as “false kava”) is sometimes mistakenly or intentionally used as an alternative botanical source for “kava” extracts. The false kava extracts do not contain active kavalactones but contain safrole, which is hepatotoxic. It is important to verify the component botanical materials in order to evaluate the quality and safety attributes of a potential botanical drug. Some studies have evaluated genetic variation in Piper sp. using the chloroplast regions matK, rbcL, rpoC1 and trnH-psbA and the nuclear ITS2 markers. However, none has focused on the identification of P. methysticum using DNA barcodes. In the present investigation, the ITS2 DNA barcode region from the nuclear genome was tested to confirm the identification and authentication of kava-kava samples. Methods: Seven P. methysticum samples were collected from three different geographic lo-cations and two P. auritum samples were collected and the ITS2 region from the nuclear genome, was amplified, sequenced and aligned to determine their genetic distances. Results: The ITS2 locus showed high amplification and sequence output with a discriminating barcode gap. A distance-based phylogenetic tree and BLAST confirmation (using blastn) revealed the ITS2 locus as a diagnostic DNA barcode for the accurate identification of kava-kava species. Discussion: In conclusion, the ITS2 region proves to be an effective and reliable DNA barcode for distinguishing P. methysticum from closely related species such as P. auritum. Its application can significantly improve the safety, quality, and traceability of kava-containing products, addressing a critical need in the standardization of botanical drugs.
Background/Objectives: Exposure of cells to ionizing radiation induces isolated DNA lesions, including single-strand breaks, apurinic/apyrimidinic sites, and oxidized bases, as well as clustered damages of different complexity. The latter types of damage are difficult to repair, and the failure to process them accurately and efficiently is related to the induction of mutagenesis, genomic instability, cancer, and aging. Since various types of clustered lesions may occur simultaneously after radiation exposure, leading to a complex architecture of DNA damage, the study of the concomitant formation and the removal kinetics of clustered DNA damage is important to determine the mutagenic and, consequently, the carcinogenic potential of ionizing radiation. Methods: With the aim of capturing real-time coexisting lesion types and assessing the repair kinetics of clustered damages, the simultaneous determination of double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters induced by gamma-irradiation of Saccharomyces cerevisiae yeast cells was performed immediately after exposure and at time intervals during incubation in Liquid Holding Recovery conditions. Results: Ionizing radiation induced lethal and mutagenic events, leading to a dose-dependent linear increase in double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters. The kinetic study showed that double-strand break frequencies declined during Liquid Holding Recovery, although a transient increase was detected at early time points. At 160 Gy, apurinic/apyrimidinic site clusters repair was evident, whereas at 400 Gy the frequency of damage increased before returning to the initial value at 24 h. In contrast, oxypurine clusters showed no net increase in repaired lesions over 24 h. Conclusions: The complex nature and topological characteristics of ionizing radiation-induced clustered DNA damage may influence lesion processing. Also, ionizing radiation may disrupt redox cellular homeostasis, leading to DNA damage and delayed effects.
Background/Objectives: Chromosome research is essential for advancing our understanding of cytogenetics, gene regulation and numerous aspects of organismal health. Staining chromosomes with 4 ',6-diamidino-2-phenylindole (DAPI) and applying Fluorescence Lifetime Imaging Microscopy (FLIM) enables the assessment of structural changes in pericentromeric and heterochromatin-rich region of chromosomes 1, with a shorter fluorescence lifetime (FLT) in the pericentromeric regions compared to the arms. Methods: We used FLIM to optimise sample preparation conditions for more robust imaging and furthermore to measure the impact of low-dose X-ray ionising radiation on chromosome structure when labelled with DAPI. Results: We applied this method to different DNA stains bound to chromosomes where only DAPI led to a clear FLT difference between the chromosome arms (p,q) with 2.98 +/- 0.12 ns and 2.65 +/- 0.07 ns at the pericentromeric region, while similar stains, such as Hoechst 33258 and NucBlueTM did not highlight these regions as clearly following FLIM analysis. Our data showed that chromosomes of cells irradiated with 0.1 Gy and 1 Gy did not show a significant change in FLTs (2.94 +/- 0.09 ns on the arms and 2.60 +/- 0.06 ns on the pericentromeric region) of chromosome 1. Whilst irradiation with 0.5 Gy led to a noticeable and significant reduction in FLT with 2.42 +/- 0.13 ns on the arms and 2.12 +/- 0.06 ns on the pericentromeric region of HeLa chromosomes. The same pattern could also be seen on X-ray-irradiated T-cell chromosomes. Conclusions: These findings indicate that DAPI FLT may be a useful tool to measure chromosomal structural changes and further suggests that chromosomes undergo distinct structural changes at the pericentromeric region following low-dose irradiation.
Liquid biopsy has evolved beyond its original role as a minimally invasive approach for mutation detection and is now being developed as a broader analytical framework for cancer detection, stratification, and longitudinal monitoring. Improvements in next-generation sequencing, assay chemistry, and computational analysis have increased analytical sensitivity, including in settings with low tumor fraction and very low variant allele abundance. These advances have expanded the utility of cfDNA analysis in measurable residual disease assessment and in the detection of low-abundance tumor-derived signals across multiple clinical contexts. At the same time, the field has shifted toward interpreting cfDNA as a carrier of higher-order biological information rather than solely a substrate for mutation calling. Fragmentation profiles, nucleosome positioning, and chromatin accessibility patterns derived from plasma DNA have been used to infer transcriptional and regulatory states, raising the possibility that cfDNA may capture functional tumor states not readily accessible through genotype-focused assays alone. These developments have prompted growing interest in chromatin-informed cfDNA analysis as a means of identifying pathway activity, enhancer usage, transcription factor occupancy, and potentially actionable biological dependencies. However, the translational relevance of many such inferences remains incompletely established. In this review, we examine the analytical advances underlying these approaches, assess the current evidence supporting their biological and clinical utility, and consider the extent to which cfDNA-derived regulatory inference may contribute to adaptive oncology and therapeutic decision-making.
Background/Objectives: Multiplex and point-of-care (POC) diagnostics require each probe to detect one intended target while rejecting many closely related sequences under shared room-temperature conditions. The conventional focus on mismatch count is incomplete: two alignments with the same number of matches and mismatches can have very different off-target risks depending on whether mismatches are clustered or distributed. We introduce a simple visual heuristic that scores mismatch placement rather than mismatch count alone. Methods: Effective complementary island (ECI) score retained matched continuity after subtracting one base for each mismatch- or gap-exposed edge. The score is S_ECI = Sigma_i ECI_i<^>2, and the design margin is Delta S_ECI = S_ECI (intended) - S_ECI (highest-scoring non-intended alignment by ECI). ECI is not a thermodynamic model; thermodynamics (Delta G37) is used separately to verify an adequate sensitivity floor. We retrospectively applied ECI to a fixed 39-target HPV capture-probe benchmark and to a public Affymetrix dataset contrasting clustered versus distributed mismatches at identical or near-identical mismatch counts. Results: In the HPV benchmark, ECI separated intended from off-target in 32/39 panels; Delta G37 favored the intended duplex in 31/39 panels; both layers were concordant in 36/39 panels. In the Affymetrix dataset (n = 8 probes, 2-4 mismatches), S_ECI correlated with reported log2 hybridization intensity (Pearson r = 0.92, p = 0.0014). Within the strict three-mismatch subset (n = 5), S_ECI remained correlated with intensity (r = 0.96; p = 0.010), while Delta G37 was uncorrelated (r = -0.04; p = 0.95), supporting the narrower claim that mismatch placement can affect signal even when mismatch count is fixed. Conclusions: ECI is not a replacement for thermodynamics, BLAST, target-accessibility analysis, empirical optimization, or machine-learning prediction. It adds one actionable readout: where to shift, shorten, or place a limited intentional mismatch so that intended retained continuity stays above the assay floor while the highest-scoring off-target island by ECI is fragmented. We provide a bench-ready workflow for multiplex, room-temperature, and POC probe design.
Background: Pulmonary aspergillosis remains underdiagnosed in patients with structural lung disease, as conventional methods might miss a part of cases. There are few real-world data on quantitative PCR performance outside immunocompromised populations. We evaluated the Aspergillus ELITe MGB Kit across two years of routine clinical practice at a tertiary pulmonology center. Methods: We retrospectively analyzed 492 consecutive ELITe MGB PCR tests (October 2023–September 2025) at the Marius Nasta Institute of Pneumology, Bucharest, Romania, performed on bronchoalveolar aspirate (n = 219), lavage (BAL; n = 202), and plasma (n = 65). Results were correlated with microscopy, fungal culture, imaging, and host risk factors where available. Results: Of 451 evaluable tests, 140 (31.0%) were significant, 7 (1.6%) low-level detected, and 304 (67.4%) non-significant or negative. Combined positivity was 32.6%. Aspirate outperformed BAL in positivity rate (42.8% vs. 30.8%; p ≈ 0.013); aspirate and BAL had similar median DNA loads (120 copies/mL each) but aspirate showed a longer tail toward very high loads (p < 0.001); raw median DNA loads (67 vs. 120 copies/mL) were not directly comparable between matrices due to differing matrix-specific lower limits of quantification (50 vs. 120 copies/mL, reflecting BAL dilution). Plasma positivity was 1.8%. Positivity peaked in May–June (42.5–45.5%), contrasting with the classical autumn pattern. Chronic obstructive pulmonary disease (COPD) was the dominant risk factor (OR = 3.45). PCR exclusively detected Aspergillus in the majority of cases where microscopy and culture were negative. Conclusions: The ELITe MGB kit demonstrates clinically meaningful diagnostic yield in a real-world pulmonology cohort. Aspirate consistently outperforms BAL, and the low-level detected category identifies a borderline population warranting prospective validation. The unexpected spring–summer positivity peak suggests a greater role for post-viral aspergillosis than previously recognized in this setting.
Recombinogenic DNA damage can initiate chromosomal rearrangements that can alter gene expression or accelerate cancer progression in higher eukaryotes. Thus, there is a critical need to identify genes that suppress chromosomal rearrangements and environmental exposures that promote genetic instability. Cell cycle checkpoints modulate the cell cycle so that DNA repair occurs before the replication or segregation of damaged chromosomes. Saccharomyces cerevisiae (budding yeast) RAD9 was the first cell cycle checkpoint gene identified, which initiated intensive research studies into the mechanisms of checkpoint activation and the phenotypes of checkpoint mutants. The budding yeast Rad9 protein serves as both an adaptor and scaffold that facilitates downstream effector activation to orchestrate a DNA damage response at multiple stages of the cell cycle, which facilitates double-strand break (DSB) repair by sister chromatid recombination. However, the role of RAD9 in homologous recombination and in suppressing gross chromosomal rearrangements (GCRs) is not completely understood. In this review we discuss how RAD9 can promote genome instability resulting from aberrant DNA replication intermediates, while suppressing DSB-associated rearrangements. We also discuss possible mechanisms accounting for the synergistic increase in genomic instability in double mutants defective in both RAD9 and recombinational repair. We emphasize that while there is an overlap between checkpoint and recombinational repair pathways, RAD9 and checkpoint pathways can function independently to suppress chromosomal instability. These studies thus elucidate checkpoint mechanisms that control homologous recombination between repeated sequences.
Background/Objectives: In recent years, efforts to understand obesity’s pathophysiology have focused on satiety signals in the hypothalamus and hormonal signalling in orexigenic and anorexigenic neurons. These signals, linked to hunger, satiety, and energy expenditure, are influenced by peptides that activate or suppress specific pathways. However, different phenotypes related to body composition result from mutations (allelic variants) in genes that encode these proteins, particularly peptide receptors. Specifically, the hormone receptor ghrelin (GHSR), located on the surface of orexigenic neurons, has been linked to the regulation of hunger. Additionally, the production and secretion of ghrelin, a peptide hormone produced by the stomach, may exhibit varying sensitivity in its receptor based on an individual’s nutritional status. Moreover, allelic variants of the GHSR gene may potentially lead to significant alterations in signalling provided by the GHSR receptor, resulting in modified hormone-binding phenotypes. In this context, the search for allelic variants that can account for diverse phenotypes, whether thinness or overweight/obesity, can aid in comprehending the pathway and defining new strategies for early laboratory diagnosis or target peptides for treatment. Methods: Initial mining produced 373 non-random SNPs located in missense regions. A total of 373 missense variants were initially identified in the GHSR gene. After applying a global minor allele frequency (MAF) filter of <1%, 20 rare missense variants remained. Results: These variants were subsequently analyzed using nine in silico pathogenicity prediction tools, resulting in the prioritization of eight variants predicted as deleterious by at least four algorithms. These variants were further analysed using the HOPE project web server and the SwissModel database. Conclusions: Through these analyses and future investigations into these mutations, we may gain a more comprehensive understanding of the implications of these mutations and their potential correlation with the pathophysiology of obesity.
