Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.

Atopic dermatitis (AD) is a widespread inflammatory skin condition that affects the population worldwide. Given the implication of microbiota in AD pathogenesis, we investigated whether human-derived Lactobacillus strains could modulate AD. In this study, we identified Lactobacillus crispatus KBL693 as a probiotic candidate for AD treatment. In vitro, KBL693 suppressed mast cell degranulation and IL-4 production by T cells, suggesting its ability to attenuate key type 2 immune responses. Consistent outcomes were observed in a murine AD model, where oral administration of KBL693 alleviated disease symptoms and reduced hallmark type 2 immune markers, including plasma IgE as well as IL-4, IL-5, and IL-13 levels in skin lesions. In addition to downregulating these AD-associated immune responses, KBL693 promoted regulatory T cell (Treg) expansion in mesenteric lymph nodes, indicating its potential to restore immune balance. Collectively, these findings highlight the therapeutic potential of KBL693 for AD through enhancement of Tregs and suppression of type 2 immune responses.

Dual specificity phosphatases (DUSPs), a subfamily of the protein tyrosine phosphatase (PTP) family, dephosphorylate not only phosphotyrosine but also phosphoserine and phosphothreonine residues. Beyond the 26 members of this family in humans, DUSPs represent the only type of PTPs found across a wide range of microorganisms, including bacteria, archaea, and viruses. This review presents a comprehensive structural analysis of human and microbial DUSPs. These proteins commonly share core features, such as a typical DUSP fold, shallow active site pocket, signature active site motif known as the P-loop, and conserved aspartate residue that acts as a general acid/base. However, DUSPs from diverse microorganisms also display unique structural and functional characteristics. Pseudomonas aeruginosa TpbA is the only bacterial DUSP identified to date, while a second candidate was proposed in this review. Archaeal DUSPs are hyperthermostable, contain a unique motif in their P-loops, and employ dual general acid/base residues. Poxviral DUSPs are characterized by the formation of domain-swapped homodimers. The presence of DUSPs across all domains of life and viruses, along with their low specificity for phosphorylated amino acids and structural similarity to classical PTPs, suggests that DUSPs represent the ancestral form of PTPs.

The Bacillus subtilis spore crust is an exceptionally robust proteinaceous layer that protects spores under extreme environmental conditions. Among its key components, CgeA, a glycosylation-associated protein, plays a critical role in modifying crust properties through its glycosylated moiety, enhancing spore dispersal in aqueous environments. In this study, we present the high-resolution cryo-electron microscopy structure of the core region of CgeA at 3.05 Å resolution, revealing a doughnut-like hexameric assembly. The N-terminal regions are disordered, whereas the C-terminal region forms the core of the hexamer. Although the loop containing Thr112 was not resolved in the density map, its location can be inferred from surrounding residues, suggesting that Thr112 is situated on the exposed surface of the hexamer. On the opposite face, a distinct electrostatic pattern is observed, featuring a negatively charged central pore and a positively charged outer surface. Modeling and biochemical studies with the putative glycosyltransferase CgeB provide insights into how the glycosyl group is transferred to Thr112. This study offers a molecular-level understanding of the assembly, glycosylation, and environmental adaptability of the B. subtilis spore crust, with valuable implications for controlling spore formation in industrial applications.

Nosocomial infections caused by Pseudomonas aeruginosa (P. aeruginosa) have become increasingly common, particularly among immunocompromised individuals, who experience high mortality rates and prolonged treatment durations due to the limited availability of effective therapies. In this study, we screened for anti-ExoS compounds targeting P. aeruginosa and identified pycnogenol (PYC) as a potent inhibitor of the type III secretion system (T3SS), a major virulence mechanism responsible for the translocation of effectors such as ExoS. Using ELISA, western blotting, and real-time PCR analyses in both P. aeruginosa and infected H292 cells, we found that PYC significantly reduced T3SS activity. Mechanistically, PYC suppressed the transcription of T3SS-related genes by downregulating exsA expression in P. aeruginosa. Furthermore, pretreatment with PYC attenuated the cytotoxic effects and reduced the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18), in P. aeruginosa-infected H292 cells. These effects were associated with the inhibition of NF-κB signaling and inflammasome activation. Taken together, our findings suggest that PYC may serve as a promising therapeutic candidate against P. aeruginosa infections by targeting T3SS-mediated virulence and modulating host inflammatory responses.

Yeast prion [PSI+], an amyloid form of the translation termination factor Sup35p/eRF3, causes translational stop codon readthrough by sequestering functional Sup35p. This unique phenotype may be analyzed via [PSI+]−suppressible nonsense alleles, and has greatly contributed to the advancement in yeast prion research. For comparing canonical reporters, like chromosomal ade1−14 or ade2−1, and plasmid-borne ura3−14, the de novo generation and characteristics of [PSI+] was investigated across common yeast laboratory strains (BY4741, 74D−694, and 779−6A). The results showed significant variability in [PSI+] induction frequency among strains. [PSI+] was successfully induced in BY4741 and frequently in 74D−694 (via Ade⁺ selection), but not in 779−6A. Notably, [PSI+] clones, even from identical genetic backgrounds, displayed vastly different nonsense suppression phenotypes depending on the reporter allele used; resulting in diverse growth patterns and suppression levels. Quantitative analyses revealed that prion seed counts fluctuated significantly based on the detection allele and observed phenotype. Furthermore, Sup35p aggregate visualization revealed distinct structural patterns between BY4741 and 74D−694, indicating strain-specific differences. Transferring [PIN+] prion variants from different strains into a common [psi−][pin−] background yielded similar [PSI+] inducibility and seed numbers, suggesting that the observed phenotypic and quantitative diversities of [PSI+] prions stem primarily from the interplay between the specific reporter detection system and the host strain's genetic background rather than solely from inherent differences in the initial [PIN+] prion or fundamental changes in the [PSI+] protein itself. This study underscores the crucial need to consider both the detection methodology and host genetic context for accurate prion variant characterization.

Human papillomaviruses (HPVs) cause abnormal cellular proliferation, leading to malignant or benign lesions, such as cervical cancer and warts. The genome of HPV16, the most prevalent high-risk oncogenic genotype within the Alphapapillomavirus genus, encodes two oncoproteins. One of these proteins, E7, interacts with multiple host proteins and modulates their functions through distinct pathways. The CR2 domain of HPV16 E7 was recently reported to interact with the μ2 subunit of clathrin-adaptor protein 2 (AP2-μ2), an adaptor complex involved in cargo internalization during clathrin-mediated endocytosis. In this study, to provide molecular insights into their intermolecular interactions, we determined the crystal structures of AP2-μ2 in complex with the HPV16 E7-derived peptides. Subsequent biochemical analyses revealed that this interaction is primarily maintained by the Y-x-x-Φ motif and further supported by acidic cluster residues of HPV16 E7. Finally, sequence alignment of the E7 CR2 domains from various HPV genotypes showed that the AP2-μ2-binding motif is largely conserved in Alpha-, Beta-, and Mupapillomaviruses, but not in Nu- and Gammapapillomaviruses.

CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.

Strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2, identified as coagulase-negative, Gram-stain-positive, halotolerant, non-motile coccoid bacteria, were isolated from traditional Korean soybean foods. Strains Mo2-6^T and S9 were both catalase- and oxidase-negative, whereas KG4-3^T and 50Mo3-2 were catalase-positive but oxidase-negative. The optimal growth conditions for Mo2-6^T and S9 were 30°C, 2% NaCl, and pH 7.0, while KG4-3^T and 50Mo3-2 grew best at 35°C, 2% NaCl, and pH 7.0. All strains contained menaquinone-7 as the predominant isoprenoid quinone, with anteiso-C_15:0 and iso-C_15:0 as the major cellular fatty acids (> 10%). Additionally, anteiso-C_13:0 was a major fatty acid in strain KG4-3^T. The DNA G + C contents of strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2 were 33.4%, 33.3%, 32.5%, and 32.7%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, formed distinct lineages within the genus Staphylococcus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, belonged to the same species. Meanwhile, dDDH and ANI values between strains Mo2-6^T and KG4-3^T, as well as comparisons with other Staphylococcus type strains, were below the species delineation thresholds, indicating they represent novel species. Based on phenotypic, chemotaxonomic, and molecular data, we propose strain Mo2-6^T as the type strain of Staphylococcus parequorum sp. nov. (=KACC 23685^T =JCM 37038^T) and strain KG4-3^T as the type strain of Staphylococcus halotolerans sp. nov. (=KACC 23684^T =JCM 37037^T).

Akkermansia muciniphila (AKK, A. muciniphila) fortifies the intestinal barrier, inhibits the colonization of pathogenic bacteria, and protects the host’s health. Nevertheless, the existing literature offers inadequate evidence to ascertain whether A. muciniphila can effectively treat Candida albicans (C. albicans) infections in vitro, and the underlying mechanisms remain ambiguous. This study, animal models were established through gavage with clinical isolates of C. albicans to induce gastrointestinal tract colonization and subsequent translocation infection. The models were subsequently administered A. muciniphila. We examined the analysis of 16S rRNA gene sequencing, metabolomics of colonic contents, and transcriptomics of colonic tissue. The intestinal barrier, inflammatory responses, and immune cell infiltration are analyzed. This study revealed that A. muciniphila markedly mitigated C. albicans translocation infection and modified the intestinal microbial community structure and metabolic attributes in model mice. After administering A. muciniphila to the translocation infection group, there was a notable increase in the prevalence of bacteria that produce short-chain fatty acids, including Eubacterium_F. Moreover, there was a significant increase in the levels of specific pathogens, including Faecalibaculum, Turicibacter, and Turicimonas. The study demonstrated that A. muciniphila treatment can improve the composition of intestinal microbiota and metabolites, augment the tight junctions of colonic tissue and diminish systemic inflammatory response. This presents an innovative therapeutic approach for the potential treatment of intestinal C. albicans infection using A. muciniphila.

CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.

Extracellular vesicles derived from probiotics have received considerable attention for their pivotal role in bacterial‒host communication. These nanosized, bilayer-encapsulated vesicles carry diverse bioactive molecules, such as proteins, lipids, nucleic acids, and metabolites. Currently, ample evidence has emerged that probiotic extracellular vesicles may modulate several processes of host physiological hemostasis and offer therapeutic benefits. This review examines the biogenesis, composition, and immunomodulatory functions of probiotic-derived extracellular vesicles in probiotic–host interactions, highlighting the therapeutic potential of probiotic extracellular vesicles in the diagnosis and treatment of conditions such as cancer and inflammatory bowel disease. We further summarize the techniques for the separation and purification of extracellular vesicles, providing a methodological foundation for future research and applications. Although the field of probiotic extracellular vesicle research is still in its infancy, the prospects for their application in the biomedical field are broad, potentially emerging as a novel therapeutic approach.

Two rod-shaped, Gram-positive, spore-forming, motile, and strictly anaerobic bacteria, FM7315^T and FM7330^T were isolated from Myeolchi-jeot, a traditional Korean fermented anchovy. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and genome sequences revealed that strains FM7315^T and FM7330^T represent novel species within the genus Haloimpatiens. The genome sizes of strains FM7315^T and FM7330^T were 3,052,517 bp and 4,194,114 bp, respectively, with G + C contents of 29.7 mol% and 28.0 mol%, respectively. Strain FM7315^T exhibited growth at 20–37°C, 0–2% NaCl, and pH range of 5.0–8.0, whereas strain FM7330^T grew at 25–45°C, 0–4% NaCl, and pH range of 5.0–9.0. Strain FM7315^T contains C_14:0, C_16:0, C_18:1 ω9c, Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c), and Summed Feature 8 (C_18:1 ω7c/C_18:1 ω6c) as major fatty acids, along with diphosphatidylglycerol, phosphatidylglycerol, glycolipid, two aminophospholipids, and five unidentified lipids. Strain FM7330^T contains C_16:0, C_17:1 ω8c, and C_18:1 ω9c as major fatty acids, along with diphosphatidylglycerol, two phosphatidylglycerols, four aminophospholipids, and six unidentified lipids. Based on their phenotypic, chemotaxonomic, and molecular characteristics, strains FM7315^T and FM7330^T represent two novel species of the genus Haloimpatiens, for which the names Haloimpatiens sporogenes sp. nov. (FM7315^T = KCTC 25939^T = JCM 37574^T) and Haloimpatiens myeolchijeotgali sp. nov. (FM7330^T = KCTC 25938^T = JCM 37575^T) have been proposed.

Pectin-rich biomass, derived from fruit and citrus processing waste, presents a promising yet underutilized resource for sustainable biofuel and biochemical production. Its low lignin content and high concentrations of fermentable sugars, including D-galacturonic acid, L-arabinose, and D-xylose, make it an attractive feedstock. Unlike lignocellulosic biomass, pectin-rich hydrolysates require milder pretreatment, improving sugar recovery efficiency. However, industrial strains such as Saccharomyces cerevisiae exhibit strong glucose preference, limiting the efficient co-fermentation of mixed sugars. While prior reviews have broadly addressed lignocellulosic biomass utilization, this mini-review uniquely centers on the specific metabolic challenges and opportunities associated with pectin-rich feedstocks. In addition to incorporating established strategies for the co-utilization of cellobiose and xylose, we highlight recent advances that allow S. cerevisiae to metabolize carbon sources specifically from pectin-rich biomass, such as L-arabinose and D-galacturonic acid—monomers not prevalent in traditional lignocellulosic biomass. By integrating discussions on sugar transport engineering, redox balancing, and pathway optimization, this review offers a comprehensive framework to overcome glucose repression and support efficient co-fermentation of carbon sources from conventional and pectin-rich biomass. Drawing on these advances, we outline practical strategies to enhance fermentation performance and expand the valorization of food processing residues in biomanufacturing.

The global spread of COVID-19 has underscored the urgent need for advanced tools to study emerging coronaviruses. Reverse genetics systems have become indispensable for dissecting viral gene functions, developing live-attenuated vaccine candidates, and identifying antiviral targets. In this study, we describe a robust and efficient reverse genetics platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The system is based on the assembly of a full-length infectious cDNA clone from seven overlapping fragments, each flanked by homologous sequences to facilitate seamless assembly using the Gibson assembly method. Individual cloning of each fragment into plasmids enables modular manipulation of the viral genome, allowing rapid site-directed mutagenesis by fragment exchange. Infectious recombinant virus was successfully recovered from the assembled cDNA, exhibiting uniform plaque morphology and genetic homogeneity compared to clinical isolates. Additionally, fluorescent reporter viruses were generated to enable real-time visualization of infection, and the effects of different mammalian promoters on viral rescue were evaluated. This reverse genetics platform enables efficient generation and manipulation of recombinant SARS-CoV-2, providing a valuable resource for virological research and the development of preventive and therapeutic antiviral measures.

Opportunistic fungal pathogens, responsible for over 300 million severe cases and 1.5 million deaths annually, pose a serious global health threat, especially in immunocompromised individuals. Among these, Candida albicans is a major cause of both superficial and invasive infections, which can progress to systemic candidiasis. One of the critical factors in C. albicans pathogenicity is the yeast-to-hyphal transition, which enables biofilm formation and promotes tissue invasion through the secretion of candidalysin, a cytolytic peptide toxin encoded by the ECE1 gene. In this study, metabolites produced by Streptomyces ambofaciens CJD34, isolated from soil samples, were screened for antifungal activity. Methoxy-apo-enterobactin (compound 1) was identified as a potential inhibitor of C. albicans virulence. Treatment with compound 1 significantly suppressed ECE1 expression and candidalysin production. In a murine subcutaneous infection model, topical application of compound 1 reduced subcutaneous colonization by C. albicans. Molecular docking analysis suggested that the inhibition of ECE1 expression was not mediated by direct binding to known upstream transcription factors, indicating an indirect mechanism of action. Collectively, these findings highlight compound 1 as a promising antivirulence agent targeting candidalysin-mediated pathogenicity in C. albicans.

Photodynamic therapy (PDT) is a known strategy for treating cancer; in PDT, photosensitizers are activated by light stimulation and then induce reactive oxygen species (ROS) production to damage cancer tissues. Recently evidence has shown that PDT can also be used as a novel treatment strategy to control pathogenic bacteria. In previous studies, the photosensitizer DH-I-180-3 was reported to effectively regulate multidrug-resistant Mycobacterium tuberculosis growth. Here, we confirmed the effects of DH-I-180-3 on the antibacterial activity and inflammatory response of macrophages to Salmonella. Photoactivated DH-I-180-3 regulated intracellular bacterial growth in Salmonella-infected macrophages. Moreover, DH-I-180-3 increased intracellular ROS levels in Salmonella-infected macrophages. The phosphorylation of the intracellular signaling proteins IκBα and JNK1/2 was increased in DH-I-180-3-treated Salmonella-infected macrophages. Additionally, we observed that DH-I-180-3 significantly increased the mRNA expression and protein secretion of the proinflammatory cytokine TNF-α and promoted phagosome maturation by upregulating EEA1, LAMP1, and Cathepsin D in Salmonella-infected macrophages. Overall, these results demonstrate that photoactivated DH-I-180-3 enhances the bactericidal response to intracellular bacterial infection by promoting inflammatory signaling pathways and phagosome maturation. Therefore, DH-I-180-3 has the potential to be developed into PDT for treating bacterial-infection.

Heme oxygenase-1 (HO-1) has antioxidant, anti-apoptotic, and anti-inflammatory properties. Emerging evidence shows that HO-1 also exhibits antiviral activity against severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus, hepatitis B virus, and Ebola virus. Its antiviral effects are mediated not only by its enzymatic function but also through the modulation of interferon-related pathways, thereby inhibiting viral replication. In this study, we investigated the antiviral effects of HO-1 on canine coronavirus (CCoV) and canine influenza virus (CIV) H3N2 using cell-based assays. To determine whether HO-1 suppresses CCoV and CIV, cells were treated with hemin to induce HO-1 expression. Hemin treatment successfully induced HO-1 expression in A72 and Madin-Darby canine kidney cells, resulting in the suppression of CCoV and CIV replication. The canine HO-1 gene was cloned into an expression vector and transfected into cells to achieve transient overexpression. Recombinant canine HO-1 protein was expressed in Escherichia coli and purified using an expression vector. HO-1 overexpression suppressed CCoV and CIV replication in cells. Following viral infection, treatment with purified HO-1 protein led to a reduction in viral protein levels. Therefore, both HO-1 expression and exogenous protein treatment effectively inhibited CCoV and CIV replication. Elevated HO-1 protein levels consistently reduced viral RNA and protein expression in vitro. These findings suggest that HO-1 could serve as a potential therapeutic agent for managing viral infections in dogs.

Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.

Pseudomonas aeruginosa (P. aeruginosa) is resistant to several drugs as well as antibiotics and is thus classified as multidrug resistant and extensively drug resistant. These bacteria have a secretion system called the "type 3 secretion system (T3SS)", which facilitates infection by delivering an effector protein. ExoenzymeS (ExoS) is known to induce cell death and activate caspase-1. In particular, patients infected with P. aeruginosa develop diseases associated with high mortality, such as pneumonia, because no drug targets an ExoS or T3SS. We selected natural compounds to treat T3SS-mediated pneumonia and chose alizarin, a red dye. We confirmed the effects of alizarin on T3SS by bacterial PCR and ELISA. It was confirmed that alizarin regulates ExoS by inhibiting exsA but also popD and pscF. Furthermore, in infected H292 cells, it not only attenuates inflammation by inhibiting lipopolysaccharide (LPS)-induced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 but also interferes with the level of ExoS delivered into the host and modulates caspase-1. We confirmed this result and determined that it led to decreases in proinflammatory cytokines such as Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), and Interleukin-18 (IL-18). Therefore, we suggest that alizarin is a suitable drug for treating pneumonia caused by P. aeruginosa because it helps to attenuate inflammation by regulating T3SS and NF-κB signaling.

Recently, floating membrane filter cultivation was adopted to simulate solid surface and enrich surface-adapted soil ammonia-oxidizing archaea (AOA) communities from agricultural soil, as opposed to the conventional liquid medium. Here, we conducted metagenomic sequencing to recover nitrifier bins from the floating membrane filter cultures and reveal their genomic properties. Phylogenomic analysis showed that AOA bins recovered from this study, designated FF_bin01 and FF_bin02, are affiliated with the Nitrososphaeraceae family, while the third bin, FF_bin03, is a nitrite-oxidizing bacterium affiliated with the Nitrospiraceae family. Based on the ANI/AAI analysis, FF_bin01 and FF_bin02 are identified as novel species within the genera “Candidatus Nitrosocosmicus” and Nitrososphaera, respectively, while FF_bin03 represents a novel species within the genus Nitrospira. The pan and core genome analysis for the 29 AOA genomes considered in this study revealed 5,784 orthologous clusters, out of which 653 were core orthologous clusters. Additionally, 90 unique orthologous clusters were conserved among the Nitrososphaeraceae family, suggesting their potential role in enhancing culturability and adaptation to diverse environmental conditions. Intriguingly, FF_bin01 and FF_bin02 harbor a gene encoding manganese catalase and FF_bin03 also possesses a heme catalase gene, which might enhance their growth on the floating membrane filter. Overall, the floating membrane filter cultivation has proven to be a promising approach for isolating distinct soil AOA, and further modifications to this technique could stimulate the growth of a broader range of uncultivated nitrifiers from diverse soil environments.

The escalating antibiotic resistance crisis poses a significant challenge to global public health, threatening the efficacy of current treatments and driving the emergence of multidrug-resistant pathogens. Among the various factors associated with bacterial antibiotic resistance, small regulatory RNAs (sRNAs) have emerged as pivotal post-transcriptional regulators which orchestrate bacterial adaptation to antibiotic pressure via diverse mechanisms. This review consolidates the current knowledge on sRNA-mediated mechanisms, focusing on drug uptake, drug efflux systems, lipopolysaccharides, cell wall modification, biofilm formation, and mutagenesis. Recent advances in transcriptomics and functional analyses have revealed novel sRNAs and their regulatory networks, expanding our understanding of resistance mechanisms. These findings highlight the potential of targeting sRNA-mediated pathways as an innovative therapeutic strategy to combat antibiotic resistance, and offer promising avenues for managing challenging bacterial infections.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.

Existing microbial engineering strategies—encompassing metabolic engineering, systems biology, and systems metabolic engineering—have significantly enhanced the potential of microbial cell factories as sustainable alternatives to the petrochemical industry by optimizing metabolic pathways. Recently, systems metabolic engineering, which integrates tools from synthetic biology, enzyme engineering, omics technology, and evolutionary engineering, has been successfully developed. By leveraging modern engineering strategies within the Design-Build-Test-Learn (DBTL) cycle framework, these advancements have revolutionized the biosynthesis of valuable compounds. This review highlights recent progress in the metabolic engineering of Corynebacterium glutamicum, a versatile microbial platform, achieved through various approaches from traditional metabolic engineering to advanced systems metabolic engineering, all within the DBTL cycle. A particular focus is placed C5 platform chemicals derived from L-lysine, one of the key amino acid production pathways of C. glutamicum. The development of DBTL cycle-based metabolic engineering strategies for this process is discussed.

The application of genetic code expansion has enabled the incorporation of non-canonical amino acids (ncAAs) into proteins, introducing novel functional groups and significantly broadening the scope of protein engineering. Over the past decade, this approach has extended beyond ncAAs to include non-proteinogenic monomers (npMs), such as β-amino acids and hydroxy acids. In vivo incorporation of these monomers requires maintaining orthogonality between endogenous and engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs while optimizing the use of the translational machinery. This review introduces the fundamental principles of genetic code expansion and highlights the development of orthogonal aaRS/tRNA pairs and ribosomal engineering to incorporate npMs. Despite these advancements, challenges remain in engineering aaRS/tRNA pairs to accommodate npMs, especially monomers that differ significantly from L-α-amino acids due to their incompatibility with existing translational machinery. This review also introduces recent methodologies that allow aaRSs to recognize and aminoacylate npMs without reliance on the ribosomal translation system, thereby unlocking new possibilities for synthesizing biopolymers with chemically diverse monomers.

Methane gas is recognized as a promising carbon substrate for the biosynthesis of value-added products due to its abundance and low price. Methanotrophs utilized methane as their sole source of carbon and energy, thus they can serve as efficient biocatalysts for methane bioconversion. Methanotrophs-catalyzed microbial bioconversion offer numerous advantages, compared to chemical processes. Current indirect chemical conversions of methane suffer from their energy-intensive processes and high capital expenditure. Methanotrophs can be cell factories capable of synthesizing various value-added products from methane such as methanol, organic acids, ectoine, polyhydroxyalkanoates, etc. However, the large-scale commercial implementation using methanotrophs remains a formidable challenge, primarily due to limitations in gas-liquid mass transfer and low metabolic capacity. This review explores recent advancements in methanotroph research, providing insights into their potential for enabling methane bioconversion.

Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3'-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes participate in various reactions involved in methionine and cysteine metabolism. The representative foodborne pathogen Staphylococcus aureus expresses the PLP-dependent enzyme MccB, which exhibits both cystathionine gamma-lyase (CGL) and cysteine desulfhydrase activities. In this study, we investigated the role of Ser323 in MccB, a conserved residue in many PLP-dependent enzymes in the transsulfuration pathway. Our findings reveal that Ser323 forms a hydrogen bond with the catalytic lysine in the absence of PLP, and upon internal aldimine formation, PLP-bound lysine is repositioned away from Ser323. Substituting Ser323 with alanine abolishes the enzymatic activity, similar to mutations at the catalytic lysine site. Spectroscopic analysis suggests that Ser323 is essential for the rapid formation of the internal aldimine with lysine in wild-type MccB. This study highlights the crucial role of Ser323 in catalysis, with broader implications for other PLP-dependent enzymes, and enhances our understanding of the molecular mechanisms involved in the selective control of foodborne pathogenic bacteria.

Bacteria-free reverse genetics techniques are crucial for the efficient generation of recombinant viruses, bypassing the need for labor-intensive bacterial cloning. These methods are particularly relevant for studying the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This study compared the efficiency of three bacteria-free approaches—circular polymerase extension reaction (CPER) with and without nick sealing and infectious sub-genomic amplicons (ISA)—to bacterial artificial chromosome (BAC)-based technology for rescuing SARS-CoV-2. Significant differences in viral titers following transfection were observed between methods. CPER with nick sealing generated virus titers comparable to those of the BAC-based method and 10 times higher than those of the standard CPER. In contrast, ISA demonstrated extremely low efficiency, as cytopathic effects were detected only after two passages. All rescued viruses exhibited replication kinetics consistent with those of the original strain, with no significant deviation in replication capacity. Furthermore, the utility of CPER and ISA in genetically modifying SARS-CoV-2 was demonstrated by successfully inserting the gene encoding green fluorescent protein into the genome. Overall, this study underscores the potential of bacteria-free methods, such as CPER and ISA, in advancing SARS-CoV-2 research while highlighting their significant differences in efficiency.

Synbiotics have become a new-age treatment tool for limiting the progression of metabolic dysfunction-associated steatotic liver disease; however, inclusive comparisons of various synbiotic treatments are still lacking. Here, we have explored and evaluated multiple synbiotic combinations incorporating three distinctive prebiotics, lactitol, lactulose and fructooligosaccharides. Of the synbiotic treatments evaluated, a combination of fructooligosaccharides and probiotics (FOS+Pro) exhibited superior protection against western diet-induced liver degeneration. This synbiotic (FOS+Pro) combination resulted in the lowest body weight gains, liver weights and liver/body weight ratios. The FOS+Pro synbiotic combination substantially alleviated liver histopathological markers and reduced serum AST and cholesterol levels. FOS+Pro ameliorated hepatic inflammation by lowering expression of proinflammatory markers including TNF-α, IL-1β, IL-6, and CCL2. FOS+Pro significantly improved steatosis by restricting the expression of lipid metabolic regulators (ACC1, FAS) and lipid transporters (CD36) in the liver. These findings are critical in suggesting that synbiotic treatments are capable of restraining western diet-induced metabolic dysfunction in the liver. Additionally, this study demonstrated that adding probiotic strains amplified the effectiveness of fructooligosaccharides but not all prebiotics.

Alcohol consumption can lead to the accumulation of harmful metabolites, such as acetaldehyde, contributing to various adverse health effects, including hangovers and liver damage. This study presents a comprehensive genomic and functional analysis of Leuconostoc suionicum VITA-PB2, a lactic acid bacterial strain isolated from kimchi, to elucidate its role in enhancing alcohol and acetaldehyde metabolism. Genomic characterization revealed key genes encoding alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), providing insights into the metabolic capabilities of strain VITA-PB2. Phylogenomic analyses confirmed its taxonomic classification and genetic similarity to other Leuconostoc species. Functional validation through in vitro and in vivo experiments demonstrated superior ethanol and acetaldehyde decomposition abilities of strain VITA-PB2, with significant reductions in blood ethanol and acetaldehyde levels observed in rats administered with the strain. Further analysis indicated that while hepatic ADH activity did not significantly increase; however, ALDH expression was elevated. This suggests that the microbial ADH of strain VITA-PB2 contributed to ethanol breakdown, while both microbial and host ALDH facilitated acetaldehyde detoxification. These findings highlight the potential of strain VITA-PB2 as a functional probiotic for mitigating the toxic effects of alcohol consumption.

Dengue, caused by four serotypes of dengue viruses (DENV-1 to DENV-4), is the most prevalent and widely mosquito-borne viral disease affecting humans. Dengue virus (DENV) infection has been reported in over 100 countries, and approximately half of the world's population is now at risk. The paucity of universally licensed DENV vaccines highlights the urgent need to address this public health concern. Action and attention to antibody-dependent enhancement increase the difficulty of vaccine development. With the worsening dengue fever epidemic, Dengvaxia^® (CYD-TDV) and Qdenga^® (TAK-003) have been approved for use in specific populations in affected areas. However, these vaccines do not provide a balanced immune response to all four DENV serotypes and the vaccination cannot cover all populations. There is still a need to develop a safe, broad-spectrum, and effective vaccine to address the increasing number of dengue cases worldwide. This review provides an overview of the existing DENV vaccines, as well as potential candidates for future studies on DENV vaccine development, and discusses the challenges and possible solutions in the field.

Salmonella enterica is a clinically significant oro-fecal pathogen that causes a wide variety of illnesses and can lead to epidemics. S. enterica expresses a lot of virulence factors that enhance its pathogenesis in host. For instance, S. enterica employs a type three secretion system (T3SS) to translocate a wide array of effector proteins that could change the surrounding niche ensuring suitable conditions for the thrive of Salmonella infection. Many antimicrobials have been recently introduced to overcome the annoying bacterial resistance to antibiotics. Enoxacin is member of the second-generation quinolones that possesses a considerable activity against S. enterica. The present study aimed to evaluate the effect of enoxacin at sub-minimum inhibitory concentration (sub-MIC) on S. enterica virulence capability and pathogenesis in host. Enoxacin at sub-MIC significantly diminished both Salmonella invasion and intracellular replication within the host cells. The observed inhibitory effect of enoxacin on S. enterica internalization could be attributed to its ability to interfere with translocation of the T3SS effector proteins. These results were further confirmed by the finding that enoxacin at sub-MIC down-regulated the expression of the genes encoding for T3SS-type II (T3SS-II). Moreover, enoxacin at sub-MIC lessened bacterial adhesion to abiotic surface and biofilm formation which indicates a potential anti-virulence activity. Importantly, in vivo results showed a significant ability of enoxacin to protect mice against S. enterica infection and decreased bacterial colonization within animal tissues. In nutshell, current findings shed light on an additional mechanism of enoxacin at sub-MIC by interfering with Salmonella intracellular replication. The outcomes presented herein could be further invested in conquering bacterial resistance and open the door for additional effective clinical applications.

This review explores current advancements in microbiome functional analysis enabled by next-generation sequencing technologies, which have transformed our understanding of microbial communities from mere taxonomic composition to their functional potential. We examine approaches that move beyond species identification to characterize microbial activities, interactions, and their roles in host health and disease. Genome-scale metabolic models allow for in-depth simulations of metabolic networks, enabling researchers to predict microbial metabolism, growth, and interspecies interactions in diverse environments. Additionally, computational methods for predicting metabolite profiles offer indirect insights into microbial metabolic outputs, which is crucial for identifying biomarkers and potential therapeutic targets. Functional pathway analysis tools further reveal microbial contributions to metabolic pathways, highlighting alterations in response to environmental changes and disease states. Together, these methods offer a powerful framework for understanding the complex metabolic interactions within microbial communities and their impact on host physiology. While significant progress has been made, challenges remain in the accuracy of predictive models and the completeness of reference databases, which limit the applicability of these methods in under-characterized ecosystems. The integration of these computational tools with multi-omic data holds promise for personalized approaches in precision medicine, allowing for targeted interventions that modulate the microbiome to improve health outcomes. This review highlights recent advances in microbiome functional analysis, providing a roadmap for future research and translational applications in human health and environmental microbiology.

With the advent of whole-genome sequencing, opportunities to investigate the population structure, transmission patterns, antimicrobial resistance profiles, and virulence determinants of Streptococcus pneumoniae at high resolution have been increasingly expanding. Consequently, a user-friendly bioinformatics tool is needed to automate the analysis of Streptococcus pneumoniae whole-genome sequencing data, summarize clinically relevant genomic features, and further guide treatment options. Here, we developed PneusPage, a web-based tool that integrates functions for species prediction, molecular typing, drug resistance determination, and data visualization of Streptococcus pneumoniae. To evaluate the performance of PneusPage, we analyzed 80 pneumococcal genomes with different serotypes from the Global Pneumococcal Sequencing Project and compared the results with those from another platform, PathogenWatch. We observed a high concordance between the two platforms in terms of serotypes (100% concordance rate), multilocus sequence typing (100% concordance rate), penicillin-binding protein typing (88.8% concordance rate), and the Global Pneumococcal Sequencing Clusters (98.8% concordance rate). In addition, PneusPage offers integrated analysis functions for the detection of virulence and mobile genetic elements that are not provided by previous platforms. By automating the analysis pipeline, PneusPage makes whole-genome sequencing data more accessible to non-specialist users, including microbiologists, epidemiologists, and clinicians, thereby enhancing the utility of whole-genome sequencing in both research and clinical settings. PneusPage is available at https://pneuspage.minholee.net/.