Antimicrobial resistance poses a major global health challenge, and infections caused by multidrug-resistant Gram-negative bacteria are associated with substantial morbidity and mortality. In contrast to many Gram-positive pathogens, Gram-negative bacteria combine intrinsic barriers with acquired determinants, including enzymatic drug inactivation, reduced outer membrane permeability, active efflux, and target modifications, which collectively compromise the efficacy of multiple antibiotic classes. Previous reviews have largely catalogued resistant pathogens or antimicrobial agents. This review provides a mechanism-focused overview of antimicrobial resistance in clinically important Gram-negative bacteria and explains how dominant resistance determinants translate into clinically relevant failure modes, such as delayed effective therapy, limited treatment options, and increased reliance on toxic last-line agents. Current and emerging therapeutic strategies are discussed through a mechanism-based lens, emphasizing newer β-lactam/β-lactamase inhibitor combinations and nontraditional approaches, including phages, antivirulence, and microbiome-based interventions. This review highlights the conceptual links between resistance mechanisms, clinical impact, and rational therapeutic choices and identifies priorities for future research aimed at mitigating antimicrobial-resistant Gram-negative infections.

Streptomyces are a crucial source of bioactive secondary metabolites with significant clinical applications. Recent studies of bacterial and metagenome-assembled genomes have revealed that Streptomyces harbors a substantial number of uncharacterized silent secondary metabolite biosynthetic gene clusters (BGCs). These BGCs represent a vast diversity of biosynthetic pathways for natural product synthesis, indicating significant untapped potential for discovering new metabolites. To exploit this potential, genome mining using comprehensive strategies that leverage extensive genomic databases can be conducted. By linking BGCs to their encoded products and integrating genetic manipulation techniques, researchers can greatly enhance the identification of new secondary metabolites with therapeutic relevance. In this context, we present a step-by-step guide for using the antiSMASH pipeline to identify secondary metabolite-coding BGCs within the complete genome of a novel Streptomyces strain. This protocol also outlines gene manipulation methods that can be applied to Streptomyces to activate cryptic clusters of interest and validate the functions of biosynthetic genes. By following these guidelines, researchers can pave the way for discovering and characterizing valuable natural products.

Fungal extracellular vesicles (EVs) have emerged as critical mediators of fungal physiology, virulence, and host–pathogen interactions. Since their first description in Cryptococcus neoformans, EVs have been identified in several fungal species and shown to transport a broad repertoire of bioactive cargo. Increasing evidence indicates that fungal EVs participate in multiple biological processes, including cell wall remodeling, stress adaptation, biofilm formation, antifungal resistance, and modulation of host immune responses. Recent advances in cryo-electron microscopy, multi-omics approaches, and functional genetics have substantially expanded our understanding of the molecular mechanisms governing EV biogenesis, cargo selection, and extracellular trafficking. These studies have further revealed that EV cargo loading is a highly regulated process linked to intracellular proteostasis, glycosylation, lipid homeostasis, and environmental adaptation. In parallel, the intrinsic immunogenicity and structural stability of fungal EVs have highlighted their translational potential as diagnostic biomarkers, vaccine platforms, therapeutic targets, and nanoscale delivery systems. Given the increasing global burden of invasive fungal infections, this review focuses on EVs derived from clinically relevant human fungal pathogens. We summarize recent advances in EV biogenesis, cargo regulation, their roles in pathogenesis, highlight emerging translational applications, and discuss key unresolved questions and future research directions in the field.

16S rRNA gene amplicon sequencing is the most widely used approach for characterizing microbial communities, yet analyzing such data requires navigating a fragmented landscape of bioinformatics tools with distinct installation requirements, parameter settings, and data formats. Here we present 16S-Pipeline, an open-source, web-based platform that provides a complete workflow from raw FASTQ files to publication-ready statistical analyses. 16S-Pipeline automatically detects sequencing type (paired-end, single-end, long-read), variable region, and sequencing platform (Illumina, PacBio HiFi, Nanopore), then performs quality filtering, primer trimming, amplicon sequence variant (ASV) inference via DADA2, taxonomy assignment against SILVA v138.1, phylogenetic tree construction, and optional functional prediction via PICRUSt2. Downstream analyses include alpha and beta diversity, taxonomic composition visualization, differential abundance testing using five complementary methods (ALDEx2, DESeq2, ANCOM-BC2, LinDA, MaAsLin2) with consensus reporting, and KEGG pathway mapping. Built-in NCBI SRA integration enables downloading public datasets for re-analysis and generates submission metadata spreadsheets for data deposition. The interactive web interface built on FastAPI and Plotly Dash enables researchers to perform complex microbiome analyses without command-line expertise. 16S-Pipeline is freely available at https://github.com/tatsu1207/16S-Pipeline under the MIT License.

Skin aging increases transepidermal water loss (TEWL), reduces elasticity, and perturbs the skin microbiome. Adipose tissue-derived stem cell exosomes (ASCE) show regenerative potential; however, their clinical effects on skin physiology and microbiome remain unclear. We conducted a split-face, randomized controlled trial in 16 adults aged ≥ 40 years with visible facial aging. One facial side received ultrasound-assisted transdermal delivery of a human ASCE-containing solution (HACS), whereas the other side received normal saline, at two-week intervals for three sessions. Biophysical outcomes (TEWL, stratum corneum hydration, and elasticity parameters R2/R5/R7) were assessed at baseline and week 2, 4, and 8. Wrinkles, pigmentation, and sebum levels were quantified using Mark-Vu imaging, and the Physician’s Global Aesthetic Improvement Scale (PGAIS) and patient satisfaction assessment scores were recorded. Skin swabs from ten participants were subjected to 16S rRNA and ITS1 sequencing. HACS treatment significantly reduced TEWL (p = 0.006 at week 2; p = 0.009 at week 8) and increased hydration (p < 0.001 at all time points) with a significant increase in elasticity (R2/R5/R7 values, p < 0.001). Both the PGAIS and patient satisfaction scores were significantly higher on the experimental side. Bacterial α/β-diversity remained largely unchanged, and no bacterial taxa remained significantly associated with skin parameters after FDR correction. In contrast, several fungal taxa showed significant positive associations with skin parameters after FDR correction, detectable only on the HACS-treated side. No significant adverse events were observed. HACS improved barrier function, elasticity, and aesthetic outcomes, whereas microbiome analyses suggested a modest fungal response associated with treatment-related skin changes in aging skin.

Antimicrobial resistance (AMR) poses an ongoing threat to global health, with the number of deaths directly attributable to AMR projected to rise to 8 million. One of the main reasons for the current crisis is the depletion of antibiotic candidates in clinical pipelines. To address this, more preclinical candidates must be advanced into development. However, the scientific challenges and limited economic incentives associated with antibiotic research have further aggravated the situation. Antibiotic hybrids, which combine two antibiotics with different modes of action, have emerged as a promising strategy to overcome AMR and are already being developed for clinical use. This approach takes advantage of the strong selective pressure exerted when two bactericidal agents act simultaneously. Importantly, because hybrids are administered as a single chemical entity, they may offer advantages over conventional combination therapies, such as simplified pharmacokinetics and dosing. Furthermore, since clinically validated antibiotics are used as the building blocks of hybrids, this strategy provides an efficient platform for generating new lead compounds. Recently, the concept of antibiotic hybrids has expanded beyond antibiotic–antibiotic conjugates to include the attachment of functional molecules designed to mitigate the disadvantages of the parent antibiotics. In this review, we summarize the definition of antibiotic hybrids, highlight representative compounds that have entered clinical evaluation, and discuss recent advances in their development.

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.

Protein quality control systems are increasingly recognized as a critical determinant of bacterial survival and antibiotic tolerance. Conventional antibiotics predominantly target nucleic acids, protein synthesis, or cell wall synthesis, yet bacterial adaptation and resistance emergence remain major challenges. Targeting the bacterial protein quality control machineries including molecular chaperones and proteases offers a promising strategy to overcome these limitations. Recent advances include small molecules and adaptor/degron mimetics that modulate the activities of chaperones and proteases, aggregation-prone peptides (APPs) that induce proteotoxic stress, and bacterial PROTAC (BacPROTAC) strategies that redirect endogenous proteases. Notably, persister and viable-but-non-culturable (VBNC) cells, which tolerate conventional antibiotics, remain susceptible to proteostasis-targeted approaches, thereby enabling killing in both actively dividing and dormant populations. Furthermore, synergistic strategies combining chaperone inhibition or protease activation with conventional antibiotics enhance bactericidal efficacy, suggesting a potential avenue to mitigate antimicrobial resistance. This review summarizes the mechanistic basis, recent developments, and translational potential of proteostasis-centered antibacterial strategies.

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.

β-Lactam antibiotics marked the beginning of an era of effective and safe treatment for bacterial infections and remain the most widely prescribed antibacterial agents today. However, the emergence of antibiotic-resistant bacteria threatens a return to the pre-antibiotic era. In particular, bacterial expression of β-lactamases inactivating β-lactam antibiotics presents a challenge in antimicrobial therapy. While inhibitors against β-lactamases have been developed to protect the therapeutic efficacy of β-lactam antibiotics, the clinical use of β-lactamase inhibitors is constrained due to their limited inhibition spectrum and the emergence of inhibitor-resistant β-lactamase variants. As an effort to tackle this issue, here we reviewed the structural and mechanistic features of β-lactamases and their FDA-approved inhibitors. Moreover, mutations in clinically isolated β-lactamases that confer resistance against their inhibitors are compiled. The comprehensive overview offered in this review aims to support and stimulate the design of next-generation β-lactamase inhibitors for combating β-lactamase-mediated antibiotic resistance.

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.

Small regulatory RNAs (sRNAs) are short noncoding RNAs that can fine-control the expression of target genes in trans at the post-transcriptional level in prokaryotes. Since there is a big challenge in constructing gene-knockout libraries, synthetic sRNAs have attracted considerable interest in synthetic biology and metabolic engineering, as they enable targeted gene knockdown without requiring chromosomal modifications. However, the development of high-efficiency synthetic sRNAs remains a demanding task that requires careful consideration of multiple design factors. Here, we provide a detailed protocol for the design and construction of synthetic sRNAs, detailing key design principles and critical optimization factors, including scaffold selection, target mRNA binding affinity, target mRNA secondary structure, and Hfq expression levels. This strategy can be broadly applied across E. coli and other bacterial hosts to modulate gene expression, thereby supporting versatile applications in synthetic biology and metabolic engineering.

Obesity is increasingly recognized as a systemic pro-inflammatory condition that influences not only metabolic and cardiovascular health but also the development and exacerbation of cutaneous inflammatory diseases. This review examines the interplay between obesity, microbial dysbiosis, and two archetypal inflammatory skin disorders—hidradenitis suppurativa (HS) and psoriasis. We highlight how obesity-induced changes in immune signaling, gut permeability, and microbiota composition—both in the gut and the skin—contribute to cutaneous inflammation. Special emphasis is placed on shared pathways such as the Th17/IL-23 and IL-22 signaling axes, adipokine imbalance, and microbial metabolites like short-chain fatty acids and lipopolysaccharides. The review critically evaluates the current literature, distinguishing preclinical insights from clinical evidence, and underscores the potential of microbiota-targeted therapies and metabolic interventions as adjunctive treatment strategies. By integrating metabolic, immunologic, and microbiome data, we synthesize emerging evidence to better understand the gut–skin–obesity interplay and guide future therapeutic innovations.

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.

The escalating threat of antimicrobial resistance has renewed global interest in peptide-based antibiotics as adaptable and effective alternatives to conventional small molecules. Peptides possess diverse mechanisms of action, high target specificity, and structural flexibility, which collectively limit the emergence of resistance. This review outlines recent advances spanning the discovery, optimization, and application of peptide antibiotics, from their biological origins and structural classifications to emerging strategies involving artificial intelligence, synthetic biology, and modern delivery technologies. Peptide antibiotics can be categorized by origin as natural, semi-synthetic, or fully synthetic, and further organized by structural class such as α-helical, β-sheet, cyclic, and extended forms. They are also grouped by function into membrane-targeted and non-membrane-targeted types. These classification schemes are not only descriptive but also critical for understanding the therapeutic potential of peptides, as each category presents distinct advantages and engineering challenges that influence stability, specificity, and overall clinical performance. Advances in artificial intelligence, synthetic biology, and continuous manufacturing are reshaping how peptide drugs are designed and produced, while innovations in drug delivery systems are addressing critical issues of stability and bioavailability. Together, these developments are laying the foundation for a new generation of peptide-based therapeutics capable of meeting the evolving challenges of antimicrobial resistance.

Protein solubility is a critical factor in the production of recombinant proteins, which are widely used in various industries, including pharmaceuticals, diagnostics, and biotechnology. Predicting protein solubility remains a challenging task due to the complexity of protein structures and the multitude of factors influencing solubility. Recent advances in computational methods, particularly those based on machine learning, have provided powerful tools for predicting protein solubility, thereby reducing the need for extensive experimental trials. This review provides an overview of current computational approaches to predict protein solubility. We discuss the datasets, features, and algorithms employed in these models. The review aims to bridge the gap between computational predictions and experimental validations, fostering the development of more accurate and reliable solubility prediction models that can significantly enhance recombinant protein production.

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.

Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathogen that causes severe community-acquired infections; however, the immune mechanisms controlling intracellular hvKp have yet to be clearly defined. In this study, we investigated the therapeutic potential of berberine against hvKp infection and elucidated the underlying molecular mechanisms using macrophage cell models and in vivo zebrafish models. Berberine significantly reduced intracellular hvKp survival in macrophages and improved survival in hvKp-infected zebrafish. Berberine markedly attenuated proinflammatory cytokine production and inhibited the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Notably, we found that hvKp exploited host lipid droplets (LD) biosynthesis to support its intracellular survival, and berberine effectively suppressed LD accumulation. Mechanistically, berberine promoted the nuclear translocation of transcription factor EB (TFEB), thereby enhancing lipolysis. Although berberine upregulated autophagy-related gene expression during hvKp infection, it did not induce lipophagy, the selective autophagic degradation of LD. Collectively, these findings indicate that berberine has potential as a therapeutic agent against hvKp infection by modulating host lipid metabolism to restrict bacterial intracellular survival.

Pathogenic Escherichia coli is a major cause of foodborne illness worldwide and includes strains capable of causing severe disease. To establish a genome-informed framework for foodborne outbreak surveillance, we analyzed 1,029 E. coli isolates from clinical, food, livestock, and environmental sources using whole-genome sequencing. Pathogenic isolates obtained from human clinical cases or linked to documented outbreaks were classified as epidemiologically defined high-risk (EpiHR), whereas the remaining pathogenic isolates were classified as non-EpiHR. Virulence-associated genomic features were extracted using a bioinformatics pipeline, and four machine learning (ML) algorithms, including gradient boosting machine, random forest (RF), and support vector machines with linear and radial basis function kernels, were evaluated. Among them, the RF model showed the best performance, achieving an area under the curve (AUC) of 0.98 and accuracy of 0.93 in 10-fold cross-validation. Additional leave-one-group-out validation showed retained discrimination across held-out sequence types and serotypes, although performance was reduced when isolates were grouped by isolation source. Evaluation using an independent test dataset of 1,908 publicly available pathogenic E. coli genomes showed an AUC of 0.97 and a sensitivity of 0.98. Feature importance analysis using Shapley additive explanations identified influential predictive features, including traT, etpB, and enterotoxin-associated genes. A reduced 10-feature model achieved an AUC of 0.79 in the independent test dataset, supporting its exploratory use for future simplified screening approaches. These results indicate that genome-based ML provides a sensitive framework for surveillance-oriented prioritization of EpiHR pathogenic E. coli isolates, with model predictions interpreted together with epidemiological information.

The widespread use of antibiotics in aquaculture has led to the emergence of multidrug-resistant pathogens and environmental concerns, highlighting the need for sustainable, eco-friendly alternatives. In this study, we isolated and characterized three novel bacteriophages from aquaculture effluents in Korean shrimp farms that target the key Vibrio pathogens, Vibrio harveyi, and Vibrio parahaemolyticus. Bacteriophages were isolated through environmental enrichment and serial purification using double-layer agar assays. Transmission electron microscopy revealed that the phages infecting V. harveyi, designated as vB_VhaS-MS01 and vB_VhaS-MS03, exhibited typical Siphoviridae morphology with long contractile tails and icosahedral heads, whereas the phage isolated from V. parahaemolyticus (vB_VpaP-MS02) displayed Podoviridae characteristics with an icosahedral head and short tail.

Whole-genome sequencing produced complete, circularized genomes of 81,710 bp for vB_VhaS-MS01, 81,874 bp for vB_VhaS-MS03, and 76,865 bp for vB_VpaP-MS02, each showing a modular genome organization typical of Caudoviricetes. Genomic and phylogenetic analyses based on the terminase large subunit gene revealed that although vB_VhaS-MS01 and vB_VhaS-MS03 were closely related, vB_VpaP-MS02 exhibited a distinct genomic architecture that reflects its unique morphology and host specificity. Collectively, these comparative analyses demonstrated that all three phages possess genetic sequences markedly different from those of previously reported bacteriophages, thereby establishing their novelty. One-step growth and multiplicity of infection (MOI) experiments demonstrated significant differences in replication kinetics, such as burst size and lytic efficiency, among the phages, with vB_VhaS-MS03 maintaining the most effective bacterial control, even at an MOI of 0.01. Additionally, host range assays showed that vB_VhaS-MS03 possessed a broader spectrum of activity, supporting its potential use as a stand-alone agent or key component of phage cocktails. These findings highlight the potential of region-specific phage therapy as a targeted and sustainable alternative to antibiotics for controlling Vibrio infections in aquaculture.

The convergence of synthetic biology and artificial intelligence is widening the gap between technological advancement and existing regulatory frameworks — a phenomenon known as regulatory lag. As global competition intensifies to capture a synthetic biology market projected to reach approximately $70 billion by 2030, nations are responding to this challenge through markedly different approaches. This study applies a comparative legal methodology, drawing on co-evolutionary theory and the Law 1.0/2.0/3.0 framework, to analyze the regulatory responses of the United States, United Kingdom, Japan, and South Korea. The analysis reveals that national differences stem less from institutional design choices than from the triggers that initiate regulatory change, yielding four distinct co-evolutionary pathways. In the United States, a shift in the judicial environment prompted regulatory adaptation; in the United Kingdom, a political transition created the conditions for legislative reform; and in Japan, administrative reinterpretation of existing rules enabled regulatory adjustment. South Korea represents a fundamentally different case. By enacting the world’s first standalone synthetic biology legislation prior to full technological maturation, Korea has pursued a top-down co-evolutionary model in which institutional design precedes and shapes the trajectory of technological development. Three conclusions emerge from this analysis: there is no single optimal co-evolutionary pathway; institutional flexibility determines the sustainability of co-evolution; and co-evolution can itself be an object of proactive design. For Korea's preemptive model to prove effective, it requires a sufficient scientific foundation, institutional flexibility, and coherence with existing regulatory frameworks — conditions that offer a reference point for other nations confronting similar governance challenges.

Microbiome studies require multiple analytical steps after initial sequence processing. These steps commonly include data harmonization, preprocessing, taxonomic profiling, diversity analysis, differential abundance testing, predictive modeling, network inference, and preparation of publication-ready outputs. Although robust packages are available for many of these tasks, routine use often depends on command-line workflows, repeated data reformatting, and method-specific scripting. These requirements can limit accessibility for experimental researchers and complicate consistent analysis across interdisciplinary teams. We developed SimpleMicrobiome, a web-based R Shiny platform that integrates established microbiome analysis methods into a single interactive downstream workflow. The application accepts standard abundance, taxonomy, and metadata tables, supports interactive preprocessing and sample filtering, and provides modules for taxa profile visualization, alpha and beta diversity analysis, ANCOM-BC2 and MaAsLin2 differential abundance testing, Random Forest modeling with SHAP-based interpretation, microbial association network inference using SparCC and SPIEC-EASI through NetCoMi, correlation heatmaps, and dbRDA/CAP-style association biplots. The platform is implemented as a modular Shiny application so that preprocessing choices are propagated across downstream analyses, results can be exported as figures and tables, and the same application can be run through the public server, source-code installation, or a Docker image.

SimpleMicrobiome consolidates major downstream microbiome analysis tasks in an accessible browser-based environment while retaining links to established analytical frameworks. The platform may reduce technical barriers for non-programming users, improve consistency across exploratory and reporting-oriented analyses, and support collaborative microbiome research. The public application is available at https://simplemicrobiome.mglab.org, the source code is available at https://github.com/yjcho2252/SimpleMicrobiome, and a Docker image for local deployment is available at https://hub.docker.com/r/mglab2252/simplemicrobiome.

While shotgun metagenomics is often used to profile antibiotic resistome in gut microbial communities, few studies have investigated if the choice of sequencing platform and assembly strategy affect what mobile genetic elements and antimicrobial resistance genes are recovered. In this study, we compared three platforms (Illumina, Oxford Nanopore, and PacBio HiFi) and seven assembly strategies on gut metagenomes from cattle, pig, and human as case studies. Long-read assemblies recovered 5- to 7-fold more plasmid sequence than Illumina in cattle and pig (mean 17.0 Mb vs. 3.1 Mb), while Illumina performed comparably in the less diverse human gut where high per-species coverage enabled effective short-read plasmid assembly. Long reads also detected more resistance genes on plasmid contigs. Hybrid assembly results depended on the algorithm: scaffolding-based OPERA-MS preserved long-read contiguity and recovered more plasmid-borne resistance genes, while the short-read-centric metaSPAdes hybrid mode produced fragmented assemblies. After collapsing haplotype redundancy, PacBio HiFi identified 2 and 49 unique multi-drug resistance plasmid lineages in cattle and pig, respectively. On the other hand, only 2 and 4 were identified from Illumina. Long reads also placed far more ARGs in a putative mobilization context (50–73%) compared to 14–21% for short reads. Platform and assembly strategy are thus key variables in mobilome and resistome characterization and should be accounted for in antimicrobial resistance surveillance.

Mycobacterium tuberculosis (Mtb) encounters diverse and fluctuating microenvironments during infection, including changes in nutrient availability and pH. While adaptation to individual host-associated conditions has been extensively studied, the impact of repeated environmental fluctuations on bacterial physiology and antibiotic survival remains unclear. In this study, we investigated how long-term adaptation to stable or fluctuating environments influences Mtb growth and drug responses. Mtb populations were serially passaged for six consecutive passages under combinations of different carbon sources and pH conditions that were either maintained consistently or altered across passages. Environmental history significantly affected bacterial growth dynamics and antibiotic survival. Notably, under identical final condition with cholesterol as a sole carbon source, populations adapted to a stable environment exhibited higher survival following bedaquiline and rifampicin treatment than populations exposed to fluctuating environments. These findings suggest that stable environments promote the optimization of growth and stress-response programs, whereas environmental fluctuations limit such optimization despite potentially increasing phenotypic heterogeneity and a possibility of survival. Together, our results identify environmental history as an important determinant of antibiotic survival in Mtb and highlight the need to consider dynamic host-like environments when investigating tuberculosis physiology and drug responses.

Triterpenoids are natural products widely found in the plant kingdom and have various pharmacological effects such as anti-inflammatory, antioxidant and anti-tumour. However, the content of triterpenoids in medicinal plants is low, and it is difficult to purify and isolate them due to their complex structure. The efficient production of some triterpenoids in chassis organisms has been achieved by constructing a heterologous triterpenoid synthesis pathway in engineered strains such as yeast, modifying the key enzymes in the pathway, and adjusting the metabolism of yeast. Modification of key enzymes in the synthetic pathway is currently an effective strategy to enhance the heterologous synthesis of triterpenoids. This paper reviews the current research progress on the modification of key enzymes downstream in the synthetic pathway and the design of key enzymes around them to enhance triterpenoid production in five main areas: 1) increasing the supply of triterpenoid precursors; 2) inhibition of the natural sterol pathway; 3) fusion expression of related enzymes; 4) compartmentalisation of the metabolic pathway; and 5) tapping and enhancing the triterpenoid efflux pump. Finally, recent advances and applications of artificial intelligence (AI) in enzyme engineering and pathway design for triterpenoid biosynthesis are highlighted. Challenges and perspectives for further increasing the yield of triterpenoid synthesis in Saccharomyces cerevisiae are presented.

Antibiotic resistance poses a serious challenge to public health worldwide; however, the development of new antibiotic classes for combating bacterial infections, especially those caused by Gram-negative pathogens, has slowed in recent years. Dual-acting hybrid antibiotics with a metabolically non-cleavable covalent bond represent an emerging strategy for developing novel antibiotic classes to overcome antibiotic resistance. The covalent connection between two antibiotics results in a fixed pharmacokinetic profile of a single molecule and can impede bacterial efflux. However, as most antibiotics do not have membrane-destabilizing activity, the resulting increase in molecular weight by connection of two antibiotics could limit their activity against Gram-negative bacteria, whose outer membrane forms a strong barrier blocking the penetration of high-molecular weight antibiotics. Here, we review recent developments in dual-acting hybrid antibiotics targeting Gram-negative bacteria, with a focus on their antibacterial efficacy. We also discuss combination therapy strategies in which the underlying molecular mechanisms of synergy have been characterized. Finally, we outline future directions for the rational design of hybrid antibiotics against Gram-negative pathogens.

Human adenovirus type 55 (HAdV-55) is an emerging respiratory pathogen associated with severe pneumonia, for which no licensed vaccines are currently available. The lack of physiologically relevant small-animal models has limited preclinical evaluation of vaccine candidates. In this study, we generated a transgenic mouse model co-expressing human CD46 and desmoglein-2 (DSG-2), key entry receptors for HAdV-55, and evaluated its utility for vaccine efficacy testing. A bicistronic expression system enabling simultaneous expression of CD46 and DSG-2 was constructed and functionally validated in vitro, demonstrating enhanced susceptibility to HAdV-55 infection. The transgenic mice exhibited dose-dependent weight loss, robust viral replication in lung tissues, and characteristic histopathological changes following intranasal challenge, recapitulating key features of human adenoviral pneumonia. Using this model, we assessed the immunogenicity and protective efficacy of an inactivated HAdV-55 (iHAdV-55) vaccine formulated with alum. Vaccination induced strong HAdV-55-specific IgG and neutralizing antibody responses, which increased over time following prime–boost immunization. Upon viral challenge, vaccinated mice showed significantly reduced weight loss and accelerated recovery compared to controls. Viral load analysis demonstrated effective control of viral replication and clearance in vaccinated animals. Collectively, these findings establish the CD46/DSG-2 transgenic mouse as a physiologically relevant and translationally valuable model for HAdV-55 infection and demonstrate that iHAdV-55 vaccination confers robust humoral immunity and protective efficacy. This platform provides a critical tool for the development and preclinical evaluation of adenovirus-targeted vaccines and therapeutics.

Recent advances in sequencing technologies, particularly long-read platforms, have substantially improved contiguity of bacterial genome assemblies and enabled the routine generation of near-complete or circular genomes. However, achieving a contiguous assembly does not necessarily guarantee accuracy. Assembly errors, including structural misassemblies, collapsed repeats, incorrect circularization, plasmid reconstruction errors, and nucleotide-level inaccuracies, remain prevalent and may lead to misleading biological interpretations if not properly identified. In this review, we provide a comprehensive overview of bacterial genome assembly from a validation-centered perspective and examine the underlying causes of draft genome formation and assembly uncertainty, highlighting the roles of repetitive genomic structures, platform-specific error profiles, and algorithmic limitations. We further emphasize that the central challenge in contemporary bacterial genomics is no longer simply to maximize assembly contiguity, but to determine whether apparently complete genomes are truly correct and sufficiently reliable for their intended downstream applications. We propose a practical decision-making framework that links sequencing strategy, assembly workflow, polishing, and validation rigor, and introduce a tiered confidence classification to guide the interpretation of genome assembly reliability. As bacterial genome sequencing becomes increasingly routine and large-scale, future efforts should prioritize accuracy, reproducibility, transparent reporting, and evidence-supported validation over completeness alone.

Plastic waste in marine environments provides novel habitats for diverse organisms, forming distinct microbial ecosystems known as the ‘plastisphere’. Although bacteria of the plastisphere have been widely studied, the role of fungi in plastisphere formation and plastic degradation remains largely unexplored. Thus, we investigated temporal changes in culturable fungal community composition on three common plastic types—high-density polyethylene, low-density polyethylene, and polypropylene—across the early (seven days) and mature (30 days) plastisphere developmental stages through a marine mesocosm experiment. In total, 436 fungal strains were isolated and identified as belonging to 179 taxa, with Penicillium, Cladosporium, Trichoderma, Aspergillus, and Fusarium as the dominant genera. Temporal shifts in the species richness of the dominant genera were observed: Cladosporium showed higher species richness at the early stage, whereas that of Trichoderma increased at the mature stage. Plastic degradation assays revealed that 54.6% of the strains exhibited measurable degradation capacity, with patterns varying by plastic type rather than fungal developmental stage. Scanning electron microscope observations revealed surface damage patterns including cracks, pitting, and erosion on the plastic surfaces. These findings provide novel insights into the composition and functional heterogeneity of culturable fungal communities in the marine plastisphere and suggest that plastisphere fungi play diverse ecological roles beyond direct plastic degradation.

The increasing environmental concerns regarding conventional plastics have led to a growing demand for sustainable alternatives, such as biodegradable plastics. Yeast cell factories, specifically Saccharomyces cerevisiae and Yarrowia lipolytica, have emerged as promising platforms for bioplastic production due to their scalability, robustness, and ease of manipulation. This review highlights synthetic biology approaches aimed at developing yeast cell factories to produce key biodegradable plastics, including polylactic acid (PLA), polyhydroxyalkanoates (PHAs), and poly (butylene adipate-co-terephthalate) (PBAT). We explore recent advancements in engineered yeast strains that utilize various synthetic biology strategies, such as the incorporation of new genetic elements at the gene, pathway, and cellular system levels. The combined efforts of metabolic engineering, protein engineering, and adaptive evolution have enhanced strain efficiency and maximized product yields. Additionally, this review addresses the importance of integrating computational tools and machine learning into the Design-Build-Test-Learn cycle for strain development. This integration aims to facilitate strain development while minimizing effort and maximizing performance. However, challenges remain in improving strain robustness and scaling up industrial production processes. By combining advanced synthetic biology techniques with computational approaches, yeast cell factories hold significant potential for the sustainable and scalable production of bioplastics, thus contributing to a greener bioeconomy.

The global rise in obesity and its associated metabolic complications underscores the urgent need for safe and effective interventions. This study investigated the anti-obesity efficacy of a probiotic mixture containing Bifidobacterium breve BR3 and Lactiplantibacillus plantarum LP3 in C57BL/6 mice with high-fat diet (HFD)-induced obesity. After obesity was established by feeding a 60% kcal HFD, the probiotic mixture was administered orally for 4 weeks. Compared with the control group, mice receiving the L. plantarum LP3 and B. breve BR3 mixture exhibited significant reductions in body weight and total fat mass, as assessed by Dual-energy X-ray Absorptiometry (DXA) and Echo Magnetic Resonance Imaging (EchoMRI). The probiotic treatment also lowered serum Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), and glucose levels, and attenuated lipid accumulation in both hepatic and epididymal adipose tissues. Transcriptomic profiling revealed upregulation of lipolytic genes (Sirt1, Pparα) and downregulation of lipogenic genes (Srebp1c, Fas), suggesting that the probiotic mixture promotes lipid catabolism while suppressing lipid synthesis. Additionally, serum adipokine levels were favorably modulated, indicating improved metabolic homeostasis. Gut microbiota analysis demonstrated an increased relative abundance of beneficial genera, including Akkermansia and Bacteroides, highlighting a microbiome-mediated contribution to the observed metabolic benefits. Overall, our findings indicate that the combined administration of Lactiplantibacillus plantarum LP3 and Bifidobacterium breve BR3 exerts multi-faceted anti-obesity effects by enhancing lipolysis, regulating lipid metabolism, and restoring a healthy gut microbial balance. This probiotic mixture represents a promising therapeutic approach for managing obesity and related metabolic disorders.

This study compared the rhizosphere microbial communities of two closely related Monotropastrum species (M. humile, Mh; and M. humile var. glaberrima, Mhg) and identified key soil factors associated with their assembly. Bacterial and fungal communities were profiled by Illumina high-throughput sequencing, and soil physicochemical properties were assessed across multiple sites in Zhejiang Province, China. The bacterial communities of both species were dominated by Proteobacteria and Acidobacteriota at the phylum level, while the dominant fungal groups belonged to Ascomycota and Basidiomycota. The two plants shared several dominant bacterial genera, including Serratia, Burkholderia-Caballeronia-Paraburkholderia, and Bradyrhizobium, as well as common dominant fungal genera such as Saitozyma and Podila. Despite these similarities, species-specific enrichment patterns were observed. The rhizosphere of Mhg contained higher abundances of Acidothermus and Lactarius, whereas Mh preferentially enriched Cedecea, Klebsiella, and Russula. Bacterial communities were shaped by pH, soil organic matter (SOM), available potassium (AK), and available phosphorus (AP), whereas fungal communities were primarily influenced by pH, alkali-hydrolyzable nitrogen (AN), and SOM (p < 0.05). These results suggest that both host identity and soil properties contribute to rhizosphere microbial assembly, with clear host-associated differentiation in microbial communities. Notably, the identified host-associated microbial taxa, particularly key mycorrhizal fungi, may serve as potential microbial inoculants, providing new opportunities for the conservation and cultivation of mycoheterotrophic plants.

Two novel bacterial strains, designated CJ20^T and CJ99^T, belonging to the genus Sphingomonas, were isolated from the Han River in South Korea and a wetland in South Korea, respectively. Cells of both strains were Gram-stain-negative, aerobic, non-motile and yellow-pigmented. Strains were shown to grow optimally at 30˚C and pH 7 in the absence of NaCl on tryptic soy medium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CJ20^T and CJ99^T belonged to the genus Sphingomonas and were most closely related to S. asaccharolytica Y-345^T and Sphingomonas koreensis JSS26^T with 97.87% and 97.58% 16S rRNA gene sequence similarities, respectively. Average nucleotide identity and digital DNA-DNA hybridization values of strain CJ20^T with S. asaccharolytica Y-345^T were 74.1% and 15.9%, respectively and those values of strain CJ99^T with S. koreensis JSS26^T were 73.9% and 15.6%, respectively. Both strains contained ubiquinone (Q-10) as the predominant respiratory quinone. The major polar lipids of strains CJ20^T and CJ99^T comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and sphingoglycolipid. The predominant fatty acids of both strains were summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c) and C_16:0. Based on polyphasic taxonomic analyses, strains CJ20^T and CJ99^T represent novel species of the genus Sphingomonas, for which names Sphingomonas degradans sp. nov. and Sphingomonas paludis are proposed, respectively. The type strains are CJ20^T (= KACC 23909 = JCM 37720) and CJ99^T (= KACC 24077 = JCM 37956).

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.

Fecal microbiota transplantation (FMT) has been successfully applied on clinical aspects, but its clinical outcomes remain unpredictable due to inconsistent donor screening protocols across hospitals, institutions, and countries. Hence, a retrospective analysis of metagenomic data from published studies on FMT donors via a unified bioinformatics workflow might contribute to the understanding of the safety considerations for donor screening and the fecal microbial profiles of qualified donors. In this study, we reanalyzed metagenomic data of 475 screened donor fecal samples from 24 studies spanning China, the USA, Canada, New Zealand, and the Netherlands. The genomic safety risks were evaluated by profiling antibiotic resistance genes (ARGs) and virulence factors (VFs), the results of which showed that no major toxin-associated virulence genes, such as Shiga toxin, Shiga-like toxin, or botulinum neurotoxin (BoNTs) genes harbored in the detected Escherichia coli, Clostridium butyricum, and Streptococcus pneumoniae, but several high-risk ARGs remained insufficiently addressed. The distribution of ARG-harboring bacteria in eligible FMT donors was country-specific. The alpha-diversity and microbial community structure were comparable between donor fecal samples from China and the USA. Interestingly, the core microbiome in fecal samples from Canada, the Netherlands, and New Zealand formed a single guild, while that from China and the USA formed two guilds, with predominantly positive intra-guild and negative inter-guild correlations, indicating that the co-abundance patterns of the core microbiome were conserved among certain countries. Furthermore, an exploratory retrospective classifier was developed based on core microbiome profiles to distinguish eligible FMT donors from general healthy individuals. These results provide evidence for integrating metagenomic sequencing into future FMT donor screening strategies.

Truncal acne significantly impairs quality of life yet remains underexplored relative to facial acne, particularly with respect to fungal ecology. The trunk represents a distinct cutaneous niche characterized by thicker epidermis, larger follicular units, and frequent occlusion, and harbors a high abundance of Malassezia species. In this study, we used internal transcribed spacer 2 (ITS2) amplicon sequencing to characterize the truncal mycobiome in patients with acne and in healthy controls and to compare fungal community features across doxycycline exposure groups. Although serial sampling was planned, seven participants contributed a single follow-up sample after doxycycline treatment, and only two participants contributed multiple follow-up samples sufficient for true within-subject longitudinal analyses; therefore, most analyses represent exposure-stratified cross-sectional comparisons rather than confirmed temporal change. At baseline, truncal acne lesions exhibited increased fungal richness and distinct community composition compared with controls. Acne lesions were more frequently enriched for Malassezia globosa, whereas healthy controls were dominated by M. sympodialis. Across doxycycline exposure groups, fungal communities remained Malassezia-dominant with substantial inter-individual variability. Doxycycline exposure was associated with partial and heterogeneous differences in Malassezia species composition without uniform normalization toward control profiles. Because only fungal sequencing was performed, bacterial–fungal interactions were inferred from prior literature and not directly measured. These findings indicate that truncal acne is associated with a distinct fungal community structure and highlight the need for integrated, longitudinal multi-omics studies to clarify treatment-associated microbial dynamics.

The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.

Basal cell carcinoma (BCC) is the most common form of skin cancer, with ultraviolet radiation recognized as the primary environmental driver; however, the potential contribution of alterations in the skin microbiota remains incompletely understood, particularly in Asian populations. This exploratory pilot study describes bacterial community patterns in BCC lesions compared with contralateral clinically normal skin in 20 Korean patients. Lesional and contralateral samples were obtained using paired skin swabs and punch biopsies and analyzed by full-length 16S rRNA gene sequencing, with targeted quantitative PCR (qPCR) of the roxP antioxidant gene of Cutibacterium acnes. Given the low-biomass nature of skin samples and the exploratory design, analyses focused on descriptive trends rather than confirmatory inference. Across available samples, C. acnes was the dominant taxon, with a trend toward lower relative abundance in BCC lesions, particularly in biopsy-derived datasets. Microbial evenness appeared higher in lesions than controls. Predictive functional profiling suggested reduced representation of vitamin B6 metabolism pathways in lesions, while qPCR analysis of swab samples showed a trend toward lower roxP/16S rRNA ratios in BCC-associated microbiota. These findings should be interpreted cautiously in light of methodological constraints, including sample heterogeneity, lidocaine exposure prior to biopsy, absence of sequencing-based negative controls, and reliance on predictive functional inference. Overall, this pilot study highlights potential differences in skin bacterial community structure between BCC lesions and contralateral skin in a Korean cohort. Larger, methodologically optimized studies incorporating metagenomic and functional validation will be required to determine whether these microbiota shifts contribute to, or result from, BCC-associated changes in the cutaneous environment.

The COVID-19 pandemic highlighted the critical role of reliable molecular diagnostics in outbreak response and the vulnerabilities of existing systems to delays and reagent instability. Armored RNA technology, which packages RNA within bacteriophage-derived capsids, offers a robust solution by combining nuclease resistance, safety, and versatility into a single platform. Armored RNA has become a trusted internal and external control for RT-qPCR and RT-LAMP, enabling accurate detection across a wide range of viral pathogens. Also, recent advances in alternative expression systems, such as plant-based and cell-free platforms, as well as the use of more stable scaffolds from bacteriophage Qβ, are enhancing yield, stability, and accessibility of armored RNA. Engineering innovations, including capsid polymorphism and optimized downstream purification, further improve efficiency and broaden possible applications. Looking ahead, armored RNA holds promise not only as a diagnostic standard but also as a delivery vehicle for vaccines and therapeutics. Encapsulation of self-amplifying RNA, small interfering RNA, or microRNA could open new pathways for rapid-response vaccines and targeted therapies, aligning this technology with the future of precision medicine. By uniting stability, scalability, and adaptability, armored RNA represents a critical component of global health preparedness, with the potential to strengthen diagnostic resilience and accelerate biomedical countermeasures in future pandemics.

Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.

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.

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.

I53-50 is a computationally designed, self-assembling protein nanoparticle (NP) that forms a stable icosahedral structure composed of 120 protein subunits coordinated through precise interfacial interactions. Through unique intelligent regulation, I53-50 exhibits sensitivity to environmental signals and display multimodal “nano-smart” properties. I53-50 has a variety of modifiable surface-active sites, which facilitates precise chemical modification, gene fusion, tag coupling, and other functionalizations, thereby promoting effective lymphatic uptake and optimizing the immune response. I53-50 NPs show great potential in vaccine development, drug delivery, and biomaterials, representing a model fusion of computational biology and nanomedicine and offering a versatile tool for precision medicine.

Antarctic fungi can effectively adapt to extreme environments, which leads to the production of unique bioactive compounds. Studies on the discovery of fungi in the diverse environments of Antarctica and their potential applications are increasing, yet remain limited. In this study, fungi were isolated from various substrates on the Fildes Peninsula in Antarctica and screened for their antibiosis activity against two significant plant pathogenic fungi, Botrytis cinerea and Fusarium culmorum. Phylogenetic analysis using multiple genetic markers revealed that the isolated Antarctic fungal strains are diverse, some of which are novel, emphasizing the underexplored biodiversity of Antarctic fungi. These findings suggest that these fungi have potential for the development of new antifungal agents that can be applied in agriculture to manage fungal plant pathogens. Furthermore, the antibiosis activities of the isolated Antarctic fungi were evaluated using a dual-culture assay. The results indicated that several strains from the genera Cyathicula, Penicillium, and Pseudeurotium significantly inhibited pathogen growth, with Penicillium pancosmium showing the highest inhibitory activity against Botrytis cinerea. Similarly, Aspergillus and Tolypocladium strains exhibited strong antagonistic effects against Fusarium culmorum. This study enhances our understanding of Antarctic fungal diversity and highlights its potential for biotechnological applications.

Collagenase and keratinase are two important proteolytic enzymes with recognized applications in biotechnology and medicine, particularly in the enzymatic removal of necrotic tissue and the control of infection. In the present work, a soil isolate of Bacillus subtilis strain AB2 (PX453297.1) was optimized for enzyme production under different nutritional and physicochemical conditions. The enzymes were recovered by ammonium sulphate precipitation and dialysis, examined by SDS-PAGE and zymography, and further assessed for pH and temperature optima, stability, the influence of metal ions, and kinetic parameters. Maximum collagenase activity (4.41 ± 0.22 U/ml) was observed at 37°C and pH 7.5 in a glucose–peptone medium, whereas keratinase production was enhanced between 37 and 40°C at pH 7.5 in lactose–peptone medium. Protein bands of approximately 55 and 33 kDa were detected, representing 6.2- and 5.5-fold purification. Collagenase showed an alkaline optimum (pH 10.0, 37–45°C) with K_m 0.31% and V_max 1.92 U/ml, while keratinase exhibited dual optima (pH 3.0 and ~7.0) with K_m 0.27% and V_max 0.84 U/ml. Biofilm assays revealed that collagenase reduced pre-formed biomass by 62–68% and viable counts by 1.1–1.7 log₁₀, clearly outperforming keratinase (41–57%, 0.7–1.2 log₁₀). When combined with conventional antibiotics, both enzymes potentiated activity, with notable synergy between collagenase and oxacillin against Staphylococcus aureus (FICI 0.31–0.37), ciprofloxacin against Pseudomonas aeruginosa (FICI 0.37–0.50), and meropenem against Klebsiella pneumoniae (FICI 0.28–0.44). These results indicate that B. subtilis AB2 produces collagenase and keratinase with distinct biochemical characteristics and strong antibiofilm properties, underscoring their promise as adjuncts in chronic wound care as well as in industrial applications.

The global rise of multidrug-resistant bacteria poses a critical threat to public health, and bacteriophage-derived endolysins have emerged as promising alternatives to conventional antibiotics. The engineered endolysin LNT113, derived from the Escherichia coli phage PBEC131 endolysin EC340, exhibits potent lytic activity against Gram-negative bacteria. This study investigated the transcriptomic responses of E. coli to sublethal LNT113 stress and identified genetic determinants required for bacterial adaptation to endolysin-induced stress. Transcriptomic analysis identified 552 differentially expressed genes (DEGs) following sublethal LNT113 exposure. Thirteen DEGs associated with stress response and envelope maintenance were individually deleted to generate thirteen mutant strains and to functionally evaluate their roles in bacterial stress tolerance. Among these, the ΔfabB and Δ(prmB–yfcL) mutants exhibited significantly reduced survival under sublethal LNT113 exposure, indicating increased susceptibility to the endolysin. Regarding the prmB–yfcL operon, individual genes were deleted to determine the gene critical for bacterial tolerance. Deletion of aroC and mepA rendered E. coli more susceptible to LNT113. Furthermore, 1-N-phenylnaphthylamine uptake assays demonstrated increased membrane permeability in the ΔfabB, ΔaroC, and ΔmepA mutants. Complementation with pWSK129::fabB, pWSK129::aroC, and pWSK129::mepA restored membrane integrity in the respective mutant strains. These findings suggest that fabB-mediated unsaturated fatty acid biosynthesis and mepA-dependent peptidoglycan remodeling are critical for maintaining envelope integrity under endolysin stress, whereas aroC may indirectly support bacterial tolerance to LNT113 via metabolic adaptation. This study provides insights into bacterial responses to LNT113 and offers a foundation for optimizing endolysin-based therapeutic strategies.

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.

Tella is a traditional beverage widely accepted by consumers, despite the lack of product consistency owing to its reliance on natural fermentation. This study aimed to identify potential industrial lactic acid bacteria (LAB) starter cultures based on their technological properties. Seven LAB strains isolated from Tella were characterized for their carbohydrate utilization, salt content, temperature, and acid tolerances, growth and acidification rates, and metabolite profiles. Most strains efficiently utilized various carbohydrates, with Lactiplantibacillus plantarum TDM41 showing exceptional versatility. The strains exhibited similar growth characteristics. Principal component analysis of stress tolerance properties revealed that L. plantarum TDM41, Pediococcus pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 exhibited superior tolerance ability. Strong acidification properties were detected in the L. plantarum TDM41, P. pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 strains after 24 h incubation at 30°C. L. plantarum TDM41 displayed the fastest acidification rate throughout the analysis period. All LAB strains produced significant amounts of diverse organic acids, including lactic acid, citric acid, acetic acid, malic acid, and succinic acid, with lactic acid being the primary acid produced by each strain. Overall, strains L. plantarum TDM41 and P. pentosaceus TAA01 prove to be potential candidates for Tella industrial starter cultures and similar cereal products owing to their robust technological properties.