Search Results (176)

Polyphosphate (polyP) is an evolutionarily conserved linear polymer of orthophosphate residues with diverse functions across organisms from bacteria to mammals. In addition to its roles in phosphate and energy storage, polyP has been implicated in thrombosis, inflammation, cancer, metabolism, cytoskeletal regulation, and neurodegenerative diseases. PolyP also acts as a molecular scaffold interacting with lysine-rich proteins and may contribute to protein folding and amyloid formation. However, biochemical heterogeneity, including variation in chain-length, subcellular localisation, and supramolecular organisation together with the absence of clearly defined mammalian biosynthetic pathways, has limited mechanistic understanding of polyP biology. Reliable detection and quantification remain challenging because current methods often suffer from limited specificity, chain length bias, insufficient quantitative robustness, and interference from other highly anionic biomolecules such as DNA or RNA. This review summarises current methodologies for polyP detection and characterisation. Emerging polyP-specific probes, including recombinant polyP-binding domains derived from polyphosphatases and conserved histidine α-helical domains may improve qualitative and quantitative analysis of polyP in complex biological systems. Improved analytical strategies will be essential to define the physiological roles of polyP and evaluate its potential as a biomarker and therapeutic target. Full article

Gut-associated lymph nodes (GALNs) constitute an important anatomical reservoir during early and chronic human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, but the fate of this compartment during terminal AIDS remains unclear. This study investigated viral persistence and lymph node injury in a rhesus macaque with long-term SIVmac239 infection and end-stage AIDS, characterized by profound CD4+ T cell depletion (30 cells/μL) and persistent viremia (1192 copies/mL). At necropsy, GALNs, including mesenteric, paracolic, and ileocecal lymph nodes, and non-gut-associated lymph nodes (NGALNs), including hepatic hilar, common iliac, and inguinal lymph nodes, were collected for viral RNA/DNA quantification, histopathology, immunofluorescence, and transcriptomics. SIV DNA was markedly lower in GALNs than in NGALNs, whereas SIV RNA was detectable only in NGALNs. GALNs exhibited extensive fibrotic remodeling, paracortical collapse, severe CD4+ T cell loss across B cell and paracortical regions, and pronounced CD8+ T cell accumulation within B cell zones. Transcriptomic profiling further revealed an exhaustion signature in GALNs, with reduced DNA replication and repair, impaired cell-cycle activity, and suppressed RNA processing, accompanied by activation of innate immune programs and attenuation of adaptive immune responses. These findings indicate that, during terminal AIDS, GALNs no longer serve as the predominant viral reservoir but instead undergo preferential and severe structural and functional exhaustion. This case highlights marked anatomical heterogeneity in lymph node vulnerability during advanced disease and supports a model in which collapse of the gut-associated immune barrier contributes to disease progression toward terminal AIDS. Full article

Rickettsia is an obligate intracellular genus of bacteria responsible for rickettsioses, a group of emerging vector-borne diseases. Their diagnosis remains challenging because of non-specific clinical manifestations, serological cross-reactivity, and low bacterial loads in clinical samples. In this study, a novel droplet digital polymerase chain reaction (ddPCR) assay was developed for the molecular detection of Rickettsia spp., and its performance was evaluated using clinical blood samples collected from dogs and cats in the Canary Islands, Spain. Primers and probes targeting the 16S ribosomal RNA (rRNA) were designed and optimized for ddPCR amplification. Analytical performance was assessed by optimising primer and probe concentrations, annealing temperature, specificity, and the limit of detection (LOD). Comparative analysis between quantitative PCR (qPCR) and ddPCR was also performed. The optimized assay employed primer and probe concentrations of 900 nM and 250 nM, respectively, with an annealing temperature of 56 °C. The assay achieved an analytical detection limit of 0.064 ng of clinical sample DNA, although sporadic positive droplets were still detectable at lower concentrations. Strong inverse correlations were observed between qPCR cycle threshold (C_T) values and ddPCR absolute quantification for both plasmid DNA (r = −0.997) and clinical sample DNA (r = −0.983). A total of 170 blood samples collected from domestic animals in the Canary Islands, with clinical suspicion of rickettsiosis, were analysed. Six canine samples (3.5%) tested positive for Rickettsia spp. The ddPCR assay developed in this study showed promising analytical performance for the molecular detection of Rickettsia spp. DNA and has the potential to become a valuable molecular tool for veterinary diagnostics and epidemiological investigations within a One Health framework. Further analytical and clinical validation studies are required before its implementation as a routine diagnostic assay. Full article

The identification of body fluids in forensic science has increasingly relied on RNA-based approaches due to their tissue specificity and ability to provide probative information beyond DNA profiling. Among the analytical platforms used for RNA detection, capillary electrophoresis (CE) has traditionally served as the standard method in forensic laboratories, offering established workflows and effective multiplexing capabilities. However, recent studies in digital PCR (dPCR) have introduced a highly sensitive alternative that enables absolute quantification of target transcripts without reliance on standard curves, particularly beneficial for low-template and degraded forensic samples. This short review provides a focused comparative evaluation of CE- and dPCR-based workflows for RNA-based body fluid identification, emphasizing analytical performance and forensic applicability. Key parameters, including sensitivity, specificity, multiplexing capacity, tolerance to degraded RNA, and interpretation methods, are critically assessed. CE remains advantageous for multiplex assay design and routine forensic implementation due to its established infrastructure and throughput. In contrast, dPCR demonstrates superior sensitivity and quantitative precision, particularly in challenging forensic samples, but is currently limited by reduced multiplexing capacity and higher costs. The review highlights the methodological strengths and limitations of both platforms and examines their suitability for forensic casework. Overall, CE and dPCR are not positioned as competing replacements at the moment but rather as complementary technologies, with their combined or context-dependent use offering improved robustness in RNA-based forensic body fluid identification. Further validation and standardization studies are required to support broader implementation of dPCR in routine forensic workflows. Full article

Detecting a KIT D816V mutation in patients with systemic mastocytosis (SM) needs a high-sensitivity assay. Additionally, high mutant KIT transcriptional activity and multilineage involvement are strong unfavorable prognostic parameters in SM. The current frequently used quantitative PCR-based KIT D816V assays have important analytical limitations, including considerable analytical variation in the low-concentration range, a need for a calibration curve, less multiplexing capabilities, and higher susceptibility to interference by PCR inhibitors. We developed, validated, and explored the diagnostic relevance of a novel multiplex KIT D816V cDNA droplet digital PCR (ddPCR), using ABL1 and KIT as reference genes. Furthermore, we correlated the expressed allele burden (EAB) with variant allele frequency (VAF) and percentage of neoplastic mast cells (nMC%) in bone marrow samples of 79 SM patients. Our assay shows very low background signals and high assay precision, leading to a linear dynamic range spanning five orders of magnitude and very low limits of detection and quantification of 0.062% (EAB/ABL1) and 0.027% (EAB/KIT). The assay detects median 57 times more mutant cDNA copies than mutant gDNA copies in SM patients and positivity in 83% of SM-suspected patients with negative gDNA ddPCR results. Furthermore, correlations between EAB, VAF, and nMC% suggest the presence of KIT D816V in non-MCs, either with or without transcriptional activity. Using our novel high-sensitivity mRNA-based multiplex ddPCR KIT D816V assay returns high diagnostic yield in patients with suspicion for SM. We recommend incorporating our assay in routine SM diagnostics to determine low-burden KIT D816V. Full article

Background: Quantitative PCR measurement of BCR::ABL1 is essential for monitoring molecular response and detecting relapse in chronic myeloid leukemia (CML) patients. Given the availability of multiple commercial kits for cDNA synthesis and minimal residual disease assessment, analytical accuracy and reliability are critical. This study evaluated two commercial RT and qPCR kits for BCR::ABL1 quantification and fusion transcript variant identification. Methods: Total RNA was isolated from peripheral blood, bone marrow, and external quality control samples from the UK NEQAS for Leucocyte Immunophenotyping program. cDNA synthesis was performed using two kits: AffinityScript (ASK) and RT Kit (RTK). BCR::ABL1 transcript levels were determined using the ipsogen^® BCR-ABL1 Mbcr IS-MMR Kit (IPS) and the LightMix^® bcr-abl t(9;22) M/m/µ Kit (TMB). Results were compared with UK NEQAS LI reference data. Fusion transcript variants were analyzed using nested PCR and a commercial qPCR assay. Results: Substantial variability was observed between the TMB and IPS assays, with moderate, non-significant correlation and wide limits of agreement. Established discrepancies resulted in different classifications of molecular response, and IPS results showed better concordance with external quality assessment data. ABL1 quantification revealed significantly higher copy numbers with the RTK compared to the ASK (p < 0.0001), enabling more reliable assessment of deep molecular responses. Statistical analyses indicated systematic and proportional bias between the methods. For variant detection, nested PCR demonstrated higher specificity, while the commercial assay showed limited discriminatory capability. Conclusions: Significant methodological differences may affect clinical interpretation, underscoring the importance of validation and standardization in CML molecular monitoring. Full article

The cashmere fibers produced by the secondary hair follicles of cashmere goats are precious textile raw materials. The secondary hair follicles exhibit a distinct annual cyclical pattern, comprising anagen, catagen, and telogen phases. Although a large amount of skin transcriptome data is currently available for cashmere goats, this study aims to systematically investigate the regulatory factors controlling the cyclical dynamics of secondary hair follicles through re-analysis of these data. Skin transcriptome data of Inner Mongolian cashmere goats were downloaded from the Sequence Read Archive (SRA) database. Bioinformatics analyses, including quality control, read mapping, quantification, differential expression analysis, Gene Ontology (GO) enrichment analysis, and KEGG pathway analysis, were performed to identify key regulatory genes governing the secondary hair follicle cycle. The results revealed a total of 1232 differentially expressed genes (DEGs) from comparisons across different phases. KEGG pathway analysis identified three signaling pathways associated with cashmere development: the cAMP signaling pathway (chx04024), the relaxin signaling pathway (chx04926), and the estrogen signaling pathway (chx04915). GO analysis yielded 335 terms, of which 236 were statistically significant (P-adj < 0.05). The DEGs were predominantly involved in biological processes such as regulation of transcription by RNA polymerase II, proteolysis, and positive regulation of transcription by RNA polymerase II. Significantly enriched cellular components included the plasma membrane, membrane, and extracellular space, and molecular functions were mainly related to protein binding, identical protein binding, and DNA-binding transcription factor activity, RNA polymerase II-specific. Based on their expression patterns, five key genes were selected: KRT25, KRT39, MAPK12, SPP1, and TCHHL1. These genes displayed significantly distinct expression profiles across the hair follicle cycle. TCHHL1 exhibited the highest expression in anagen, intermediate expression in catagen, and the lowest expression in telogen, with significant differences among all three phases. MAPK12 was specifically upregulated in telogen and was significantly more highly expressed than in both anagen and catagen. SPP1 showed high expression in anagen and catagen but extremely low expression in telogen. KRT39 displayed significantly higher expression in both anagen and catagen compared to telogen. KRT25 reached its highest expression in catagen, which was significantly different from that in telogen, while its expression in anagen showed no significant difference from either telogen or catagen. This study characterized the regulatory genes of the secondary hair follicle cycle in cashmere goats at the transcriptional level and analyzed the association between their expression patterns and cycle regulation. Full article

This study established long-term wastewater surveillance of Aichi virus (AiV) in Maryland. AiV, a member of the Kobuvirus genus associated with acute gastroenteritis, has established itself as an integral marker for wastewater-based monitoring; however, two key research questions remain unaddressed for the Baltimore metropolitan area: (1) whether AiV is consistently detectable in municipal wastewater throughout the year, and (2) whether its concentrations exhibit a measurable seasonal pattern. To address these hypotheses, influent samples were collected on a weekly basis from WWTP-A and WWTP-B from January to December 2023 (with grab sampling conducted at WWTP-A and automated collection deployed for the influent sampling of the water treatment plant B). All samples (n = 51) were subjected to PEG 8000 concentration, RNA extraction, cDNA synthesis, and RT-qPCR quantification. We observed AiV RNA in 94.12% of the samples from both facilities (25/51 at WWTP-A and 23/51 at WWTP-B) with concentrations that ranged from 2.5 to 3.63 log₁₀ gc/L and a seasonal pattern showing consistent declines: loads for WWTP-A declining from winter (3.58 log₁₀ gc/L) to fall (2.56) and for WWTP-B from winter (3.28 log₁₀ gc/L) to fall (2.31). The year-round constant AiV presence provides a strong basis for its use as a stable viral marker within wastewater-based epidemiology efforts. Full article

Background: The m⁷G cap structure, which mimics the natural cap of eukaryotic mRNA, is a critical determinant of mRNA vaccine efficacy, safety, and stability. However, its precise quantification remains challenging due to complex impurity profiles and the high physicochemical similarity between the target cap and related impurities. Although liquid chromatography mass spectrometry (LC-MS) is widely employed for this purpose, current methodologies still face significant limitations, including labor-intensive sample preparation, low analytical throughput, poor reproducibility in quantifying low-level impurities, and a lack of universally applicable strategies across diverse mRNA vaccine platforms. Methods: We systematically optimized sample preparation and LC-MS detection workflows. RNase H-mediated cleavage was compared with DNAzymes, guide DNA probes were rationally designed, and thermostable RNase H was introduced for one-step denaturation and cleavage. To establish an accurate, efficient, and universal sample preparation workflow. Chromatographic conditions were optimized using an ion-pairing reagent system to suppress ESI-MS metal adducts. Eliminating sample purification improves recovery, reduces manual handling errors, and boosts assay efficiency. Results: Through optimally designed guide DNA probes, RNase H cleavage specificity reached ≥98% with high cleavage efficiency, offering higher efficiency than DNAzyme. Furthermore, the incorporation of thermostable RNase H enabled a single-step workflow combining high-temperature denaturation and site-specific cleavage, substantially streamlining sample preparation. On the chromatographic side, optimization of the ion-pairing reagent system effectively suppressed metal adduct formation in electrospray ionization mass spectrometry (ESI-MS). This advancement enabled direct injection of the 5′ cap fragments without purification, achieving high-recovery quantification while demonstrating broad compatibility across mainstream LC-MS platforms. The optimized assay reduces the total analytical workflow from 4~6 h to under 1.5 h. Conclusions: Combining high accuracy, robustness, and broad platform compatibility, this method offers a universal, high-throughput analytical solution for mRNA vaccine quality control and continuous process development. Full article

Background/Objectives: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine-metabolic disorder. Rotterdam-defined phenotypes may need adjunctive, non-invasive signals for research-level phenotypic stratification. Saliva deserves attention because it is easy to collect repeatedly, acceptable in outpatient settings, and may reflect oral-systemic inflammatory and endocrine-metabolic interactions. This study evaluated whether salivary qPCR signals for Lactobacillus, Prevotella, and Bifidobacterium differ across Rotterdam-defined PCOS phenotypes and controls, with emphasis on the exploratory relevance of Prevotella. Methods: This cross-sectional proof-of-concept study included 110 women: 87 with PCOS and 23 controls. PCOS phenotypes were classified according to the Rotterdam criteria. Salivary microbial targets were assessed using SYBR Green-based genus-specific qPCR. Available instrument-export spreadsheets were reviewed for standard-curve quality control. Because the available assay outputs did not support robust absolute quantification, inferential analyses used Cq-based microbial signals only. Lower Cq values indicate stronger target DNA signal. No universal bacterial 16S rRNA reference, exogenous spike-in, salivary flow correction, or fully validated copy-number conversion was available. Group differences were evaluated using non-parametric tests with Bonferroni-corrected post-hoc comparisons. ROC, regression, and correlation analyses were retained as hypothesis-generating analyses only. Results: Prevotella Cq values differed significantly across groups (p < 0.001), with lower median Cq values in phenotypes A, B, and C than in phenotype D and controls. Lactobacillus Cq values did not differ significantly (p = 0.249). Bifidobacterium showed an overall group difference (p < 0.001), but its pattern and assay performance were less consistent. Among women with PCOS, Prevotella Cq values were associated with Ferriman-Gallwey score and polycystic ovarian morphology. Conclusions: Salivary Prevotella showed the clearest exploratory genus-level qPCR signal across Rotterdam-defined PCOS phenotypes. The findings support further technical and clinical validation of saliva-based microbial profiling as a possible adjunct to conventional PCOS phenotyping. They do not validate Prevotella as a standalone diagnostic biomarker, do not define clinical cutoffs, and do not quantify absolute bacterial load. Full article

Whole-genome sequencing (WGS) is central to outbreak response for high-consequence ribonucleic acid (RNA) viruses, but useful genomes depend on workflow design, sample quality, biosafety, diagnostic breadth, infrastructure, and bioinformatics as much as sequencing platform. The 2026 Bundibugyo virus disease outbreak in the Democratic Republic of the Congo and Uganda provides a case example. Bundibugyo virus (BDBV) was already known from the 2007–2008 Uganda and 2012 Democratic Republic of the Congo outbreaks, but sparse historical genome sampling, Ebola virus-centered diagnostic assumptions, non-specific febrile and viral hemorrhagic fever presentations, and difficult field conditions created a need for broad differential diagnosis, rapid species assignment, and representative genome generation. This review compares outbreak WGS strategies by the degree of prior viral sequence knowledge required, distinguishing direct RNA sequencing, random-primed complementary DNA (cDNA) sequencing, sequence-independent amplified cDNA sequencing, background-depleted or particle-enriched cDNA sequencing, probe-based hybrid capture, tiled amplicon sequencing, portable field sequencing, culture-derived sequencing, and associated bioinformatics workflows. For BDBV-like outbreaks, the most defensible strategy is staged and adaptive: broad viral hemorrhagic fever and febrile illness differential testing at recognition; Filoviridae-wide testing when filovirus disease remains plausible; divergence-tolerant first-genome recovery; quantification-cycle-informed sequencing prioritization without BDBV-only diagnostic narrowing; validated amplicon scale-up; and representative sequencing across locations and time. Full article

Long-read sequencing technologies provide portable and flexible service, making them attractive for small-scale sequencing studies. However, many existing RNA-sequencing analysis frameworks are designed for transcriptome-wide analyses and require substantial computational resources. Here we present a lightweight and reproducible computational pipeline for targeted long-read transcriptomic profiling using Oxford Nanopore Technologies (ONT) cDNA sequencing data. The pipeline was evaluated using targeted long-read transcriptomic datasets generated from formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma samples previously classified as microsatellite instability—high (MSI-high) by PCR-based testing. Libraries were sequenced on the Oxford Nanopore MinION platform using R10.4.1 flow cells. Application of the workflow enabled rapid quantification of mismatch repair gene expression and detection of immune-related transcripts including CD8A, PDCD1, and HAVCR2 across multiplexed barcode samples. The pipeline performs targeted alignment of long-read sequencing data to a custom transcript reference panel using minimap2, followed by gene-level read counting and normalization using reads-per-million (RPM). Optional modules enable immune marker profiling, detection of reads aligning to multiple genes, exploratory variant analysis, and visualization of expression patterns. By combining simplicity, reproducibility, and minimal computational overhead, the present pipeline provides an accessible framework for targeted transcriptomic analysis of long-read sequencing data. It may facilitate adoption of ONT-based transcriptomic profiling in settings with restricted computational resources. Full article

Background: Acute lung injury (ALI) is a major contributor to mortality in neonatal sepsis, yet the mechanisms underlying early lung damage remain incompletely understood. Although extracellular traps (ETs) have been implicated in inflammatory injury, the cellular origin and regulatory pathways of ET formation in neonatal sepsis remain unclear. This study aimed to determine the source of ETs and to investigate the role of hypoxia-inducible factor-1α (HIF-1α) in regulating macrophage extracellular traps (METs) formation and lung injury. Methods: Neonatal sepsis was induced in mice by intraperitoneal injection of cecal slurry. METs formation was assessed by immunofluorescence staining, Western blotting, and extracellular DNA quantification. Selective depletion of macrophages or neutrophils was performed to determine the cellular source of ETs. In vitro experiments were conducted using macrophages stimulated with lipopolysaccharide or phorbol 12-myristate 13-acetate. RNA sequencing analysis and pharmacological inhibition were used to examine the roles of HIF-1α, glycolysis, and enolase 2 (ENO2) in METs formation, lung injury, and survival outcomes. Results: We identify macrophages as a predominant source of ETs in the lung and demonstrate that METs contribute to lung injury in neonatal sepsis. Depletion of macrophages or pharmacological inhibition of METs formation markedly attenuated lung injury and improved survival in neonatal sepsis mice. Mechanistically, we suggest that HIF-1α promotes METs formation by driving glycolysis in macrophages. Furthermore, this process appears to involve the upregulation of key glycolytic enzymes, including ENO2, potentially facilitating METs release. In turn, METs are implicated in enhancing macrophage inflammatory activation, which could exacerbate lung injury. Importantly, pharmacological targeting of HIF-1α pathways reduces METs formation, attenuates lung inflammation, and improves survival outcomes. Conclusions: These findings suggest a role for HIF-1α in regulating METs formation and support that targeting this pathway could represent a potential therapeutic strategy for neonatal sepsis-associated acute lung injury. Full article

Quantitative polymerase chain reaction (qPCR) is a widespread method to quantify RNA or DNA. The results are reported as cycle of quantification (Cq), scaled to absolute numbers of copies or relative to reference genes. The reported Cq values of the same reaction vary between different machines and cannot be compared between different laboratories. This study shows that the third derivative zero (TD0) method is machine independent and more reproducible than the classic Cq calculations. Together with the mean PCR efficiency it allows the calculation of the number of copies initially present (Ncopy), a parameter easy to interpret. A large dataset was created for the evaluation of this method including amplicons with different length, primer concentrations, reaction mixes, and fluorescence reporter systems. Furthermore, the calculated Ncopy values can be corrected at the same time using known concentrations of a standard and for the expression of reference genes and combining absolute and relative quantification. The algorithms were implemented in the open-source program RDML-Tools, which can perform all steps of a qPCR analysis using the raw fluorescence amplification data and is available on the internet. We conclude that qPCR analysis today should widen its focus and include the three essential parameters, TD0, mean PCR efficiency and Ncopy. Full article

Soil degradation remains a major challenge in sub-Saharan Africa, particularly within smallholder farming systems characterized by low-input agriculture and unsustainable land use practices. Sustainable agriculture production requires a good understanding of soil characteristics across diverse farming contexts. This study assessed soil health and microbial diversity across three contrasting systems: long-term fallow (aggregated farm A), high-input (aggregated farm B), and conventional smallholder (non-aggregated farm C) farms experiencing declining productivity. Soil samples collected from the three contrasting systems were analyzed for physicochemical properties and microbial communities using high-throughput DNA sequencing. Microbial communities were characterized by using amplicon sequencing targeting bacterial 16S rRNA and fungal ITS gene regions, allowing taxonomic profiling and inference of microbial diversity patterns. The two aggregated farms predominantly had clay soils, with pH values ranging from 6.78 to 7.39 and organic carbon content from 1.17% to 1.64%. In contrast, conventional farms had loamy to clayey soils with a pH value of 5.88 and an organic carbon content of 1.25%. Both types of aggregated farms showed moderate to high concentrations of total nitrogen (0.12–0.13%), phosphorus (38.79–151.36 mg/kg), and potassium (548.84–943.52 mg/kg), along with elevated levels of calcium and magnesium, though fertilizer application was inconsistent across the sites. Microbial diversity analysis revealed significant differences among the systems. The dominant bacterial phyla were Pseudomonadota (48.5%), Acidobacteriota (34.2%) and Actinomycetota (19.6%), while the primary fungi included Ascomycota, Basidiomycota and Mortierellomycota. Functional profiling using COG and KEGG databases showed distinct variations in microbial potentials, with a high diversity of Actinobacteria, Acidobacteria and Proteobacteria. Functional profiles inferred from amplicon-based predictions represent potential metabolic capabilities and should be interpreted cautiously as indicative rather than direct functional gene quantification. Correlation analyses between soil and microbial communities provided essential baseline data to support the development of sustainable farming practices and land restoration strategies aimed at improving soil fertility and agricultural productivity in these degraded landscapes. Full article