Airborne microbiological monitoring is increasingly relevant for identifying and quantifying viral pathogens; however, standardized analytical workflows for respiratory viruses recovered from environmental matrices remain limited. This study reports the full analytical validation of a quantitative RT-qPCR workflow for the simultaneous detection and quantification of SARS-CoV-2, influenza A, influenza B, and RSV A/B from air samples collected on gelatin membrane filters. The protocol optimizes RNA extraction and amplification from this challenging matrix and provides a standardized procedure suitable for reproducible laboratory implementation. All validation parameters-amplification efficiency, linearity, recovery, matrix interference, limit of detection (LOD), limit of quantification (LOQ), repeatability, and reproducibility-were assessed in compliance with ISO 20395 and MIQE guidelines and met all predefined acceptance criteria. The workflow showed excellent linearity (R2 ≥ 0.99 for all targets), high amplification efficiency (92-103%), robust recovery (81.98-92.28%), and no significant matrix interference (±10%). LODs ranged from 5 to 9 copies/reaction, and LOQs from 8 to 10.5 copies/reaction. Precision assessments demonstrated acceptable intra- and inter-test variability (CV% ≤ 35%, SD < 0.19). Overall, this validated and standards-compliant RT-qPCR workflow provides a reliable, sensitive, and reproducible analytical tool for laboratory quantification of airborne respiratory viruses collected on gelatin membrane filters, supporting standardized virological analysis of aerosol samples.
Fecal coliform (FC) is a key indicator of surface water contamination from animal and human waste, and elevated FC levels are associated with waterborne diseases, which remain major public health concerns. However, existing studies are often limited by sparse sampling, time-consuming laboratory analyses, and inadequate representation of complex nonlinear relationships among water quality parameters (WQPs) influencing FC dynamics. Therefore, this study aims to apply and compare machine learning models for predicting FC concentrations using physicochemical WQPs, including pH, salinity (WS), water temperature (WT), electrical conductivity (EC), total dissolved solids (TDS), dissolved oxygen (DO), turbidity (Turb), biochemical oxygen demand (BOD), and water hardness (WH), while evaluating their predictive contributions and interactions in coastal Bangladesh. FC concentrations ranged from 188 to 1620 CFU/100 mL, with a mean of 765.57 CFU/100 mL. Pearson correlation analysis ranked the predictors as Turb > BOD > DO > pH > WS > WH > WT > TDS > EC. Among the models, the multilayer perceptron (MLP) outperformed random forest regression (RFR), multiple linear regression (MLR), extreme gradient boosting (XGB), and support vector regression (SVR). During testing, MLP achieved the highest R2 (0.822), with the lowest RMSE (109.48 CFU/100 mL) and MAE (99.95 CFU/100 mL). Notably, the optimal input combination (IC-5), comprising only five WQPs (Turb, BOD, DO, pH, and WS), explained 80.8% of the variability in FC concentrations and achieved 98.30% of the R2 obtained using all nine WQPs. These findings may provide a useful basis for microbial water-quality assessment in comparable coastal environments.
The viable but non-culturable (VBNC) state of Campylobacter jejuni complicates food safety monitoring, as current assays such as propidium monoazide quantitative PCR (PMA-qPCR) rely on membrane integrity and can overestimate the viability of physiologically compromised cells. This study evaluates a dielectrophoresis (DEP)-based microfluidic method as a rapid, label-free approach for characterizing heat-stressed C. jejuni. We established selective DEP capture conditions (≥4.0 MHz) that distinguished VBNC-candidate cells (defined here as culture-negative but membrane-intact) from autoclaved dead controls. DEP-based enumeration agreed with PMA-qPCR at moderate temperatures (50-60 °C), whereas a critical decoupling emerged at elevated temperatures (≥70 °C): DEP responsiveness markedly diminished despite persistent PMA-qPCR signals, indicating a loss of electrical polarizability in cells that retained membrane impermeability. In parallel, the number of proteins identified by LC-MS/MS declined with treatment severity, with a pronounced loss of respiratory-chain and ATP-synthesis components; this pattern is consistent with, but does not directly demonstrate, a temperature-dependent decline in energy-generating capacity. Together, these results show that DEP responsiveness and membrane permeability report distinct and only partially overlapping physiological states, and that DEP enables label-free capture and recovery of the electrically responsive subpopulation. Importantly, DEP responsiveness should be interpreted as an electrical-functional phenotype rather than direct proof of cellular viability; thus, rather than serving as a stand-alone viability assay, DEP provides information complementary to membrane-permeability-based methods for characterizing heat-stressed C. jejuni.
We developed a novel single nucleotide variant (SNV)-based genotyping method for Prototheca bovis based on mitochondrial and plastid genome polymorphisms. Sequencing of five PCR amplicons targeting six informative SNVs discriminated field isolates with a Simpson's D value of 0.927, providing a simple and robust epidemiological typing method.
A two-step protocol employing Tris-equilibrated phenol (pH 4.5) and sodium dodecyl sulfate was optimized to efficiently isolate DNA-free, intact RNA from gram-negative bacteria. This method reduced processing time and reagent use, thereby minimizing RNA degradation and contamination. The extracted RNA was suitable for gene expression profiling.
Validating the ecological distribution of a newly isolated bacterial species in natural hosts remains challenging due to the lack of specific detection assays and the cost of large-scale screening. Here, we describe a dual-strategy bioinformatics pipeline that leverages publicly available 16S rRNA gene amplicon sequencing data to reliably and inexpensively confirm target bacterial presence. The method first extracts hypervariable regions from the target bacterium's full-length 16S rRNA gene and evaluates their specificity by calculating an A-value-defined as the highest sequence similarity to any non-target strain in reference databases. Regions with an A-value below the 98.7% species threshold are selected. These are then aligned against Amplicon Sequence Variants (ASVs) from public datasets to compute a B-value (highest similarity to ASVs within a sample). A novel classification logic (B > A) is applied to designate samples as positive or negative, reducing false positives. The pipeline incorporates multi-level controls, including process/biological negatives and positives. Testing with novel species (Clostridium sp. nov.) and a formally described species (Streptococcus lishijunsis), along with common commensal species demonstrated that region-specific performance varies, highlighting the need for pre-validation. The framework successfully distinguished target-positive from negative samples, with phylogenetic support for specificity. This approach provides a rigorous, cost-effective, and accessible workflow that links in vitro isolation to in vivo ecological validation using existing public data.
Recombinant human granulocyte colony-stimulating factor (rh-G-CSF) is useful in tissue regeneration due to its angiogenic and anti-inflammatory actions. The current study focused on the downstream process of A7G mutant rh-G-CSF (m-rh-G-CSF) and the effects of m-rh-G-CSF on human fibroblasts. The m-rh-G-CSF was purified and had 97.24% purity, a final protein concentration of 0.814 g/L and a molecular weight of 18,784 Da. The addition of m-rh-G-CSF significantly elevated the ATP level in the treated cells in a concentration-dependent manner. The maximal ATP level was achieved at a concentration of 2.5 μg/mL and was 89% higher than in the control cells and 13% higher than in reference rh-G-CSF. In the scratch-wound assay, 92.67% and 98.69% wound closure were observed at 24 and 48 h after the addition of m-rh-G-CSF, respectively, compared with 49.93% in control cells at 48 h. These findings support the potential of m-rh-G-CSF as a therapeutic candidate for fibroblast-mediated wound healing.
Composting, also referred to as aerobic fermentation, is a key method for recycling organic waste. The composting pile is characterized by substantial heterogeneity, which may influence microbial populations; however, the effects of this spatial heterogeneity on microbial communities have not been sufficiently investigated. In this study, we compared the microbiomes of particle-associated and composite samples collected during the thermophilic phase-the most active period for organic matter degradation. Our findings reveal marked microbial divergence between particle-associated and composite samples. Notably, anaerobic groups such as Clostridia and Deltaproteobacteria were enriched in particle samples, whereas Bacilli dominated the composite samples. In both sample types, the microbial community underwent continuous succession throughout the thermophilic stage. Specifically, composite samples shifted from early Bacilli dominance to a gradual increase in Actinobacteria in later stages, while particle samples showed a progressive rise in Clostridia, peaking at Day 6. This pattern underscores that composting is driven by both aerobic and anaerobic microbial populations. Based on bioinformatic screening, we selected three thermophilic bacterial isolates that were prevalent in both particle and composite samples and evaluated their capacity to accelerate composting. Inoculation with these isolates led to a rapid temperature rise (peaking at 74.1 °C) and a prolonged thermophilic phase. Overall, this study demonstrates that the spatial heterogeneity of composting piles creates distinct microenvironments that foster both aerobic and anaerobic microbial populations, with divergent successional trajectories between particle-associated and composite samples, and further shows that bioinformatics-driven selection of thermophilic isolates can effectively accelerate the composting process.
The escalating contamination of water resources by heavy metals, bicarbonates, seawater, and drain-wastewater intrusion is a multifaceted challenge, necessitating sustainable remediation strategies. A multi-objective bioremediation approach that acclimatizes and utilizes microalgae to mitigate pollutants concurrently is adopted. Twelve microalgae were acclimatized to heavy metals (copper, cadmium, and chromium) up to 100 mg/L, salinity similar to seawater up to 50% v/v, elevated bicarbonate levels up to 40.78 g/L, and drain-wastewater up to 50% v/v, simultaneously (sample not from an actual plant). This was carried out in batches using modified-Zarrouk's medium added with heavy metals, seawater, bicarbonate, and wastewater, with a four-step increment to reach the above concentrations and slowly acclimatize during microalgae cultivation and bioremediation. Bicarbonate in the medium was intermittently added with 3 g/L increments, gradually escalating to 13.5 g/L over a 15-day batch. Other operational conditions for mixotrophic microalgae cultivation were: illumination- red (680 nm, 45000 lx); initial sugar- 5.5 g/L; initial pH- 9.2; and temperature- 28 ± 2 °C. The microalgae demonstrated a synergistic capacity for multi-contaminant removal and production of value-added byproducts, achieving removal efficiencies exceeding 90% for heavy metals, a 100% reduction in bicarbonate levels, and a marked decrease in salinity. Lipids from Nannochloropsis sp., Arthrospira platensis, Scenedesmus obliquus, Chlorella vulgaris, Chlorococcum sp., and Navicula sp. 2 produced 3rd-generation quality biodiesel via transesterification, with high fatty acid methyl ester content and viscosity as per ASTM-D6751 standard. Eco-friendly wastewater treatment technology developed showed high bioremediation efficiency and also provided alternative energy as a sustainable solution, supporting UN-SDGs 6 and 7.
Influenza A virus (IAV) remains a major focus of global public health concern, as it can cause influenza-like illness and severe respiratory infections. Therefore, achieving accurate and sensitive detection of IAV would greatly facilitate disease diagnosis and control. The two main conventional technical methods for IAV detection that are reviewed in this article are viral nucleic acid amplification analysis and immunological assays. At the same time, this review pays special attention to the cross-integration of these two types of techniques with modern engineering technologies, and systematically introduces novel biosensors (offering significantly enhanced sensitivity) and point-of-care testing (POCT) devices (enabling rapid on-site diagnosis) developed based on these two principles. However, it is important to note that the majority of these novel platforms remain at the proof-of-concept stage, with validation largely confined to laboratory settings. By delineating the current state of these technologies, this review identifies key challenges and future directions for translating research innovations into clinical practice.
Aeromonas veronii is a major bacterial pathogen in freshwater aquaculture, yet rapid species-level quantification remains challenging within the genetically complex genus Aeromonas. We developed a singleplex hydrolysis-probe (TaqMan) quantitative PCR (qPCR) assay targeting an A. veronii-discriminatory region of the aerolysin gene (aerA) and validated it according to MIQE recommendations. Plasmid standards gave a linear range of 2 to 2 × 106 copies/reaction (R2 = 0.9962) with 100.5% amplification efficiency. The endpoint limit of detection was 2 copies per reaction, and 20 copies per reaction was set as the practical reporting limit based on reproducible detection and low intra- and inter-assay variation. Analytical specificity was evaluated with genomic DNA from an 18-strain panel, with reproducible amplification observed only for A. veronii. The assay was further tested in 55 fish-tissue and 11 aquaculture-water DNA extracts. NH8B-1D2 sample-process monitoring was used for matrix-level recovery correction, and tissue and water extraction blanks were undetermined. The aerA target was detected in all tested gill, stomach/intestine, spleen, kidney/head kidney, pond-water filter and Xiamen seawater filter extracts, and in 10/11 liver extracts. Median NH8B-corrected loads were highest in gill among tissues and higher in pond-water filters than in Xiamen seawater filters. A separate Vibrio harveyi inhibition-check assay indicated no obvious amplification-stage inhibition. This assay supports rapid quantification of aerA-positive A. veronii in fish and aquaculture-water matrices.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a high-consequence tick-borne pathogen requiring accurate molecular tools for reliable viral load assessment. This study evaluated and compared the performance of a laboratory-optimized quantitative real-time RT-PCR (qRT-PCR) assay with a digital PCR (dPCR) platform for the detection and absolute quantification of CCHFV RNA. Analytical validation was performed using a characterized viral isolate propagated in Huh7 cells, followed by serial dilution analysis to assess sensitivity, linearity, and dynamic range. Clinical performance was further evaluated using 27 human plasma samples obtained from suspected or confirmed Crimean-Congo hemorrhagic fever (CCHF) cases. All samples were analyzed in parallel using both qRT-PCR and dPCR platforms. qRT-PCR demonstrated strong linearity across a wide dynamic range (R2 = 0.992); however, amplification efficiency exceeded optimal limits (104.8%, slope: -3.211), with increased variability observed near the detection threshold. In contrast, dPCR provided absolute quantification independent of standard curves, exhibiting high analytical precision and a functional limit of detection of approximately 30 copies/μL. Clinical analysis revealed that the majority of samples possessed low viral loads (Ct ≥ 37), where qRT-PCR showed increased variability, while dPCR maintained stable quantification across all concentration ranges. Overall, a significant correlation was observed between the two methods (R2=0.7121, p<0.0001), although dPCR demonstrated enhanced sensitivity in low viral load conditions. These findings suggest that dPCR provides valuable diagnostic clarity for precise quantification of CCHFV RNA, particularly in low-titer samples near the detection threshold, highlighting its potential utility in supporting molecular diagnostics and epidemiological surveillance.
The increasing global burden of petroleum-derived plastic pollution has intensified the search for sustainable and biodegradable alternatives to conventional plastics. Polyhydroxyalkanoates (PHAs) are biodegradable microbial polyesters with considerable potential to replace petroleum-based plastics; however, research has predominantly focused on bacterial production systems, while microeukaryotic microorganisms remain largely unexplored. In this study, the ciliated protozoan Paramecium jenningsi, isolated from stagnant freshwater, was investigated as a microeukaryotic platform for PHA biosynthesis. Growth conditions were optimized using Bold Basal Medium (BBM), and intracellular PHA accumulation was initially screened using Sudan Black B and Nile Blue A staining. PHA production was induced under glucose-enriched and HgSO₄-stressed conditions, and polymers were extracted after 24, 48, and 72 h of exposure. The highest polymer yield (0.36 g/L) was obtained from glucose-treated cultures after 24 h, significantly exceeding that of HgSO₄-treated and control cultures. The recovered polymers were characterized using Fourier-transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). FTIR analysis revealed characteristic functional groups associated with PHAs, while GC-MS detected prominent 2-butenoic acid ester derivatives indicative of polyhydroxybutyrate (PHB)-related polymers. Notably, GC-MS also revealed medium- and long-chain-length hydroxyalkanoate monomers (C4-C19) alongside the short-chain-length PHB-associated derivatives, indicating that P. jenningsi produces a structurally heterogeneous PHA copolymer rather than PHB alone. These findings demonstrate the ability of P. jenningsi to accumulate PHB-like biopolymers under both carbon-rich and metal-stressed conditions and provide the first evidence supporting this species as a potential microeukaryotic host for PHA production. The study expands the diversity of microbial systems available for biopolymer research and highlights the potential of protozoan platforms for sustainable bioplastic development.
Bioactive peptides derived from probiotics are gaining widespread attention for their potential as nutraceuticals for the treatment of complex metabolic disorders. However, efficient recovery of peptide fractions with enhanced biological activity remains a significant challenge. In the present study, probiotic biomass obtained from a commercially available probiotic formulation was subjected to enzymatic hydrolysis using trypsin, followed by differential precipitation using magnesium sulfate (MgSO₄) and zinc sulfate (ZnSO₄) to enrich peptide fractions. SDS-PAGE was used to characterize the recovered peptides, and reverse-phase high-performance liquid chromatography (RP-HPLC) was used to evaluate molecular weight distributions and chromatographic profiles. ZnSO₄ precipitation yielded a higher quantity of peptide fraction (160 mg/L culture) compared with MgSO₄ precipitation (140 mg/L culture). SDS-PAGE analysis revealed enrichment of low-molecular-weight peptides predominantly within the 2-10 kDa range. In contrast, RP-HPLC analysis demonstrated differences in peptide composition and relative abundance between the two fractions. The biological activities of the isolated peptides were assessed through DPPH radical scavenging, Oil Red O staining, and MTT cytotoxicity assays. Both peptide fractions exhibited dose-dependent antioxidant, anti-lipidogenic, and cytotoxic activities; however, the ZnSO₄-precipitated fraction consistently demonstrated significantly greater activity than the MgSO₄ fraction. These findings indicate that differential salt precipitation influences peptide recovery and biological activity, with ZnSO₄ being a more effective precipitating agent under the experimental conditions used. These findings suggest that probiotic-derived peptides may serve as potential bioactive molecules for future therapeutic development targeting metabolic disorders.
Mpox has re-emerged as a significant global zoonotic threat, driven mainly by two large waves the 2022 worldwide Clade IIb outbreak and the 2024 Clade Ib epidemic in Central Africa. This review examines the challenges of interpreting this evolving virus from molecular, epidemiological, and bioinformatics perspectives, with a focus on global health workforce preparedness. Clade IIb largely moved through sexual transmission across countries, but Clade Ib has appeared in a wider population-women, children, and individuals infected through household spread without any sexual contact. Early case series suggest that Clade Ib may cause a more severe disease burden, but more research is needed to directly compare severity and fatality rates with Clade IIb due to the limited number of current studies. The review examines the virus's strategies for evading the host's immune defenses throughout its ∼197 kbp genome, including how it disrupts interferon signaling and creates decoy receptors. This review summarizes the clinical findings of PALM007 and STOMP, noting that neither trial achieved its main efficacy endpoint making routine tecovirimat use less compelling-while leaving open whether it helps particular high-risk groups. A further point is that immunity from the MVA-BN vaccine wanes with time, leading to the growing adoption of booster vaccinations. In conclusion, the review calls for a One Health approach pairing genomic tracking with ecological intelligence and including wastewater surveillance to fill existing gaps in knowledge and enhance the global handling of new orthopoxvirus threats.
Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.
Rapid screening of arginase-producing microorganisms is challenging because traditional assays often need enzyme purification, large reagent volumes, spectrophotometric equipment, or labour-intensive procedures, limiting high-throughput microbial bioprospecting. We developed a compact UV-assisted O-phthalaldehyde (OPA) fluorogenic micro-spot assay for rapid semi-quantitative detection of arginase activity in crude marine bacterial extracts. After converting L-Arginine to L-Ornithine enzymatically, the reaction mixtures were derivatized with OPA and β-mercaptoethanol, producing cyan fluorescence visible under 365 nm UV light in a micro-spot format on a parafilm surface. Fluorescence was quantified digitally in ImageJ by calculating corrected total fluorescence (CTF), thereby eliminating the need for specialised fluorescence instrumentation. Out of 83 marine bacterial isolates, 17 showed clear fluorescence signals indicating arginase activity, while negative controls had minimal background fluorescence. Validation with a conventional ninhydrin-based ornithine assay showed a strong correlation (R2 = 0.9392) between fluorescence and ornithine levels, confirming the method's semi-quantitative reliability. While it is primarily a rapid screening tool rather than a precise analytical method, its compatibility with crude extracts, low reagent use, fast workflow, and minimal equipment needs make it ideal for high-throughput microbial enzyme screening and marine bioprospecting.
The exposure to ultraviolet (UV) radiation is one of the key exogenous processes that contribute to photoaging, oxidative stress, and barrier dysfunction. Naematelia aurantialba is a very rare medicinal and edible fungus which is an organism with a large pharmacological potential, yet there is a gap in understanding the molecular processes involved in the anti-photoaging and soothing repair actions that the fermentation product is known to possess. The aim of this study was to understand the protective role of Naematelia aurantialba fermentation broth (NAF) against UV-induced skin cells damage and the possible molecular mechanisms of damage. The fermentation of Lactobacillus plantarum was used to prepare NAF. Its physicochemical characteristics were measured by use of DPPH, hydroxyl radical scavenging, and elastase inhibition and its biocompatibility by use of erythrocyte hemolysis experiment. We created UVA induced HFF-1 photoaging model, UVB induced HaCaT barrier damage model, and capsaicin induced sensitive model in order to measure cell viability and expression of factors associated with them. It was found that NAF had a large free radical scavenging capacity and elastase activity, and had a great biocompatibility over its interesting concentration (hemolysis rate < 5%). Molecularly, NAF activates mitochondrial homeostasis, boosting the gene and protein expression of the SIRT1 and SIRT3 proteins, and thereby strongly prevents the release of the enzyme, matrix metalloproteinase-1 (MMP-1) and suppressed collagenase degradation. At the same time, NAF prevents the capsaicin-induced overexpression of TRPV1 substantially preventing neurogenic inflammation, and increases aquaporin 3 (AQP3) and claudin-1 (CLDN1), mending the skin barrier. The NAF can produce various anti-photoaging, soothing, and barrier repair effects by activating the dermal SIRT1/3- MMP -1 axis, and blocking the epidermal TRP V1 pathway, which justifies its potential as an innovative functional plant raw material.
