Analysis of microorganisms in drinking water is useful for assessing water quality and potential public health risks. Although DNA-based analyses are widely used, DNA extraction methods are not standardized. Herein, the DNeasy PowerWater Kit was compared to a modified DNeasy PowerWater Kit protocol that employs a series of pretreatment steps. Water samples from two community water systems were collected. Biomass from each sample was concentrated onto separate filters, from which DNA was extracted using the original or the modified method. The modified method extracted significantly higher quantities of DNA (p < 0.01), but the concentrations of total bacterial biomass, total microbial biomass, Legionella spp., and Mycobacterium spp. obtained via quantitative PCR were statistically similar between the two methods (p ≥ 0.1). Vermamoeba vermiformis concentrations in both extracts were similar (p = 0.3) for one system but ~0.2 log gene copies/L greater using the modified method (p = 0.01) for the other system. The microbial communities differed significantly according to alpha and beta diversity metrics. However, operational taxonomic unit (OTU) comparisons suggest the differences were largely from OTUs of low abundance (i.e., <0.15% relative abundance), as relative abundances of most dominant and moderately abundant OTUs were similar. Overall, these results suggest that the modified method may not merit use in studies involving large numbers of samples because of the substantially greater labor and supplies requirements, although it provides marginal improvements in DNA recovery that may benefit studies of rare taxa.

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 × 10[6] copies per reaction (R[2] = 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-/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 than 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.

This study describes a newly designed microplate sealing lid (SLIM-V®) for susceptibility testing of vapours of plant-derived volatiles against pneumonia causing bacteria. The in vitro antibacterial effect of volatile compounds (trans-anethole, 4-isopropyl-3-methylphenol, β-thujaplicin, thymoquinone) and essential oils (EOs) from aerial part of Thymus vulgaris, fruits of Hellwigia musifolia, leaves of Wurfbainia mindanaensis, and two forms of Kolowratia eruciformis with red and white calyces were evaluated in vapour and liquid phase using broh microdilution volatilization (BMV) assay and its modification employing SLIM-V. Thymoquinone was found to be the most active against all bacteria tested (MICs 16-64 μg/mL), followed by β-thujaplicin (32-1024 μg/mL). The consistent results of both methods support validity and reliability of SLIM-V. GC-MS/FID analysis using two detectors (mass spectrometer and flame ionization detector) and two columns of different polarity (HP-5MS/dB-HeavyWAX) showed that β-caryophyllene, α-pinene and p-cymene, thymol, and 1,8-cineol are predominant compounds of H. musifolia, K. eruciformis red and white forms, T. vulgaris, and W. mindanaensis EOs, respectively. More detailed analysis of K. eruciformis using GC × GC-TOFMS confirmed variances in the chemical composition of EOs of both forms suggesting their chemotaxonomical distinction. Based on the results of antimicrobial assay, thymoquinone can be proposed for further research focused on development of inhalation therapies. In comparison with BMV assay, the experiments demonstrated that the use of SLIM-V represents a significant advancement in the methodologies assessing antimicrobial activity of volatiles. This innovative laboratory tool has potential to be safer, easier to handle, and provide more reproducible and sensitive results than other methods.

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.

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 (R[2] = 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 (R[2]=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.

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 (R[2] = 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 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.

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.

We describe a simple modification of the classical double-agar-layer (DAL) assay that integrates Robert Koch's dilution-streaking to improve spatial separation of individual plaques and streamline the isolation of purified phages. In the method, named DS-DAL (dilution-streaking-DAL), a clarified phage lysate isolated against its specific host is first dilution-streaked across the surface of hard agar plates (1.5%); then the corresponding host-containing soft agar (0.75%) overlay is poured over the air-dried streaked surface, and the plates are incubated until plaques form along the streak tracks. We applied the technique to purify host-specific coliphages and Shigella phages intended for phage therapy in poultry, as well as polyvalent phages against Aeromonas for aquaculture applications. The approach produced discrete, well-spaced plaques with undiluted samples in the second or third line of the dilution streak (depending on the initial titer values), requiring only one plate to purify the phage, compared with 7-8 plates required for that many dilutions in conventional overlay plating. Thus, the dilution-streak DAL is a low-cost assay for rapid phage purification that requires no specialized equipment and is compatible with routine downstream workflows in both research and applied settings.

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.

Bloodstream infections (BSIs) pose a serious threat to patients undergoing hematopoietic stem cell transplantation (HSCT). However, the low abundance of routine infection markers-including procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6 (IL-6)-limits their diagnostic utility for early detection. Identifying markers that rise earlier than conventional indicators remains a pressing challenge. We previously prepared a recombinant mannan-binding lectin (MBL) termed M1 and found that magnetic beads coated with M1 (M1-beads) primarily enriched abnormal leukocytes from the blood of HSCT patients with BSI within 3 h of fever onset. Compared with leukocytes from normal controls, these M1-captured cells exhibited more pronounced features of PANoptosis, including higher expression levels of gasdermin D (GSDMD), phosphorylated mixed lineage kinase domain-like protein (p-MLKL), and interleukin-18 (IL-18); more evident double staining with Annexin V-FITC/propidium iodide (PI); and more distinct morphological characteristics of PANoptosis under transmission electron microscopy (TEM). At this early time point, however, routine plasma infection markers (PCT, CRP, and IL-6) remained not significantly elevated in BSI patients. Collectively, these findings demonstrated that GSDMD and p-MLKL rose earlier and reached higher levels in M1-captured PANoptotic leukocytes, highlighting their potential as early host-derived biomarkers for the adjunctive diagnosis of BSI in HSCT patients.

Surface-layer (S-layer) proteins, forming the outermost envelope of many bacteria and archaea, exhibit extraordinary structural precision and self-assemble into two-dimensional crystalline lattices with square, hexagonal, or oblique symmetry. These monomolecular arrays, typically 5 to 25 nm in periodicity (varying by species), offer defined porosity and serve as robust biological nanoplatforms. Their innate capacity for self-assembly and molecular ordering has attracted significant attention in nanobiotechnology, vaccine development, biosensing, drug delivery, and ultrafiltration. S-layers are especially valued for their ability to mimic viral capsids, enhance antigen presentation, stabilize lipid bilayers, and provide highly organized scaffolds for enzyme immobilization and nanopatterning. Recent experimental achievements include the use of S-layer fusion proteins for mucosal vaccine delivery and the development of recombinant S-layer-based electrochemical biosensors. However, transitioning these advances to commercial-scale applications remains challenging. Limitations include the scalability of high-purity protein production, cost-effective recombinant expression, stability under harsh industrial conditions, and unresolved regulatory pathways for biologically derived nanomaterials. Additionally, synthetic alternatives present practical and economic competition. Nonetheless, interdisciplinary efforts in synthetic biology, materials science, and computational modeling are addressing these bottlenecks. Innovations such as cross-linkable domains, fusion with polymers or lipids, and predictive structure-function modeling are improving the robustness and adaptability of S-layer systems. As current research advances from theoretical potential to functional prototypes, S-layer proteins offer transformative prospects across medical, industrial, and environmental domains. This review uniquely integrates mechanistic S-layer biology with engineering-for-manufacture, protein-design workflows, and commercialization roadmaps - offering actionable protocols and benchmarks not covered in prior syntheses.