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Few weight loss interventions have isolated the use of gamification through points to incentivize the social support needed for successful weight loss during a mobile intervention. To compare a group that receives points [mLife (mobile Lifestyle Intervention for Food and Exercise)+points] vs. a group that did not receive points (mLife) for completing social support activities. Differences in social support perceptions, provision, and receipt were examined as part of a 12-month weight loss intervention. The mediating role of these activities with weight loss was also examined. Participants with overweight/obesity (n&#x2009;=&#x2009;243 enrolled) were recruited to participate in a 12-month randomized trial delivered via the mLife app. Perceived social support was measured via survey [Multidimensional Scale of Perceived Social Support (MSPSS)] and by ratings of support received in the app. Social support provision was assessed via the number of social support activities completed in the mLife app over 12&#x2009;months, and social support receipt was assessed as the number of social support activities a participant received over 12&#x2009;months. At 12&#x2009;months, mLife+points participants had greater perceived social support (difference between groups: total MSPSS 0.52&#x2009;&#xb1;&#x2009;0.16, P&#x2009;<&#x2009;.01; ratings of support via app: 0.17&#x2009;&#xb1;&#x2009;0.07, P&#x2009;=&#x2009;.02) and greater rate ratio (RR) of providing social support (RR&#x2009;=&#x2009;2.23, P&#x2009;<&#x2009;.001) and receiving social support (RR&#x2009;=&#x2009;2.4, P&#x2009;<&#x2009;.001) as compared to mLife participants. Both social support receipt and provision mediated the relationship between group and 12-month weight loss. Incentivizing social support provision during a weight loss intervention via points is a potential way to increase social support perceptions, provision, and receipt. The Clinical Trials Registration #NCT05176847. Participants who were randomized to a mobile weight loss intervention incentivizing them to provide social support to one another using points reported greater perceived social support and provided and received more social support than a nonpoints condition.

Rapid identification of bacterial species from patient samples is crucial for clinical decision-making. In severe infections, such as bloodstream infections, the early start of an effective treatment is directly associated with reduced mortality rates. Current rapid species identification methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or multiplex PCR, require specialized hardware and extensive technical support that prevents application in resource-limited settings. Here, we present a staining and imaging procedure for bacterial smears using fluorescent dyes directed against intracellular structures and cell wall components. Data on relevant features were extracted from segmented images and used to train a machine learning (ML) model for species classification. The method was tested on clinical isolates from 126 patients. For the seven most common bacteria, the classification performance, indicated by area under the receiver operating characteristic (ROC) curve, ranged from 0.8 (Klebsiella pneumoniae) to 1 (Pseudomonas aeruginosa). Species that were not part of the training dataset, were reliably classified as unknown species. These results hold promise for the identification of further species, particularly Enterobacterales, and clinical application.

The resistance&#x2012;nodulation&#x2012;division (RND) family of multidrug efflux transporters is widely distributed in Gram-negative bacteria. Although their roles in mediating antibiotic resistance have been well known, our understanding of how they are altered to augment bacterial adaptation to antibiotic selection remains at an infancy stage. Here, we report the identification of a mutation-based mechanism that empowers the function of the CmeB efflux protein, an RND-type transporter in the zoonotic pathogen Campylobacter. During our surveillance study, we identified Campylobacter isolates that were phenotypically resistant to florfenicol but lacked known florfenicol resistance mechanisms. Using natural transformation and whole genome sequencing, we first linked the phenotype to sequence polymorphisms in the cmeB and subsequently demonstrated that both the T136A and M292I mutations in CmeB are required for the resistance phenotype. The mutations elevated Campylobacter resistance to florfenicol, ciprofloxacin, and other classes of antimicrobial agents. Structural modeling and molecular dynamics simulations revealed that the two residues were localized in the drug-binding pocket of CmeB, and the T136A and M292I substitutions enhanced hydrophobic interactions, stabilized CmeB-antibiotic binding, and lessened steric hindrance in the drug-binding pocket, thereby facilitating antibiotic extrusion by CmeB. Analysis of the Campylobacter genomic sequences deposited in the NCBI database revealed that T136A- and M292I-harboring isolates were found in 35 different countries and associated with various host species, indicating the widespread distribution and clinical relevance of the two mutations. Together, these results identified a new mechanism underlying CmeB-mediated multidrug resistance and provide a potential target for clinical surveillance of antibiotic-resistant Campylobacter.

Bacteriophages and archaeal viruses are the most abundant biological entities on Earth. Through a long-standing co-evolutionary arms race, they have driven the emergence of a diverse repertoire of prokaryotic defense systems. This review summarizes these systems, highlighting their diverse antiviral mechanisms across distinct stages of viral infection, from surface barriers and inducible innate responses to specific adaptive defenses, and the intricate interplay between these defense strategies. By examining host-virus counter defense dynamics, the trade-off between survival benefit and adaptive cost, the co-evolution of RNA and protein components, and the comparison with eukaryotic immune systems, we underscore the intrinsic complexity and evolutionary plasticity of prokaryotic antiviral immunity. A deeper understanding of these processes and mechanisms will not only shed light on the origins and evolution of the immune system but also provide valuable opportunities for the development of biotechnological tools.

Mycobacterium abscessus, one of the most antimicrobial-resistant bacteria, is increasingly recognized as the cause of infections that are difficult to treat. Novel genetic manipulation tools are required to elucidate the biology, pathogenesis, and antibiotic resistance mechanisms of M. abscessus. In this study, we modified the method used to prepare M. abscessus electrocompetent cells to achieve efficient transformation, and then optimized the CRISPR-Cas9-assisted genome-editing tools to allow efficient genetic manipulation. Using these tools, we constructed 66 efflux pump mutants of M. abscessus and investigated their roles in drug resistance and virulence. We found that different efflux pumps play distinct roles in drug resistance and survival in Galleria mellonella larvae. Finally, we confirmed that MAB_2806-2807, involved in transportation of triacylglycerides, is vital for the drug resistance and virulence of M. abscessus. The molecular biology tools developed in this study will facilitate molecular research on M. abscessus. In addition, the study on efflux pumps might provide new targets for the development of new drugs and treatment regimens for M. abscessus infection.

Infectious disease diagnostics has been transformed by metagenomic next-generation sequencing (mNGS), an unbiased approach that detects bacteria, viruses, fungi, and parasites in a single assay. By sequencing all nucleic acids in a sample, mNGS overcomes the narrow detection scope and slow turnaround of conventional tests, substantially improving pathogen detection. In conditions such as meningitis/encephalitis, sepsis, and pneumonia, mNGS frequently identifies etiologies missed by routine diagnostic tests, thereby facilitating earlier pathogen-directed therapy and, in selected settings, improving clinical management and outcomes. This approach is particularly valuable for immunocompromised, pediatric, and intensive care unit (ICU) patients with atypical infections. Currently, clinical mNGS workflows primarily rely on short-read sequencing platforms (e.g., Illumina), whereas long-read platforms (e.g., Nanopore, PacBio) offer advantages for rapid or high-resolution applications. Optimized bioinformatics and stringent quality control are essential for reliable results. Beyond clinical diagnostics, mNGS provides valuable genetic data on antimicrobial resistance (AMR) and pathogen phylogeny, supporting public health and outbreak surveillance (e.g., wastewater monitoring and variant tracking). Current challenges include distinguishing colonization from infection, interpreting sequencing data quantitatively, and reducing cost and turnaround time. Looking ahead, emerging strategies such as targeted panels, rapid automated workflows, and host&#x2011;response integration are expected to further shorten time&#x2011;to&#x2011;result and improve diagnostic specificity. Parallel progress in ethical and regulatory frameworks remains essential to ensure responsible implementation. To support clinical adoption, a standardized framework for clinical interpretation of mNGS results, together with associated training, has been developed and implemented. Overall, mNGS is likely to become an increasingly important component of infectious disease diagnostics, with ongoing innovations expected to broaden its clinical and epidemiological impact.

Bacteria deploy diverse innate immune systems to combat bacteriophage infections. The cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a type of innate prokaryotic immune system. CBASS synthesizes cyclic-oligonucleotide through cGAS/DncV-like nucleotidyltransferases (CD-NTases) to activate downstream effectors, which kill bacteriophage-infected bacteria, thereby stopping phage spread. One major class of CBASS contains a homolog of eukaryotic ubiquitin-conjugating enzymes, either as an E1-E2 fusion or a single E2 enzyme. Both enzymes function by regulating CD-NTase activity. Currently, many structures of CD-NTases have been reported, but there are only a few reports of structures where CD-NTases form complexes with the associated E2. In this study, we analyzed the length and classification of the CD-NTase in two types of type II CBASS-E1E2/JAB-CBASS and E2-CBASS. We found that the CD-NTase in E2-CBASS is longer and predominantly belongs to clade G. We also present the structure of the SmCdnG-SmE2 complex with the bound GTP substrate, which indicates the conservation of the donor binding pattern. Interestingly, we discovered that SmCdnG contains a conserved C-terminal &#x3b1;-helix and &#x3b2;-sheet structure, which is uniquely involved in forming a complex with SmE2. We also found that the structure of the E2 protein in the E2-CBASS system is highly conserved. Altogether, we provide mechanistic insights into the E2-CBASS system.

The development of safe and effective radioprotective agents with minimal side effects, particularly for high-dose exposure, remains a global priority. E0703, a novel steroidal compound structurally derived from estradiol, has shown promising radioprotective efficacy with limited estrogenic activity in prior pharmacodynamic studies. In this study, E0703 was found to significantly increase the abundance of Akkermansia muciniphila (AKK) in the intestines of both irradiated and non-irradiated mice. Co-administration of E0703 and AKK markedly improved the 7-day survival rate of mice exposed to a lethal 8.5&#x2009;Gy dose of radiation. E0703 induced beneficial transcriptional changes in AKK, with enrichment in metabolic pathways such as amino acid biosynthesis, aminoacyl-tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. These alterations supported the production of glucosamine 6-phosphate (GlcN-6-P) by AKK, which contributed to intestinal tissue regeneration following irradiation. Single-cell transcriptomic analysis revealed that E0703 significantly increased the proportion of intestinal stem cells and goblet cells by Day 5 post irradiation. Mechanistically, E0703 modulated the oxidative phosphorylation pathway in these cell types, including regulation of Muc2 production. E0703 also enhanced AKK abundance in irradiated mice, particularly in the presence of mucin, thereby elevating the availability of GlcN-6-P-a critical substrate for intestinal organoid repair. These findings indicate that E0703 exerts direct effects on goblet cells and AKK, promoting host-microbe interactions that facilitate intestinal regeneration and improve survival following radiation exposure.

SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it a surrogate model for evaluating the efficacy of vaccines and drugs against SARS-CoV-2.

Microorganisms play a vital role in human health through their interactions with the body. Studies of host-microbe mechanisms and interactions are crucial for advancing health management. Recently, the organoid-based models have provided new platforms in this field. Derived from human tissues, these models offer several advantages over traditional systems and, when combined with advanced analytical techniques, they enable deeper insights into host-microbe interactions. In this review, we summarize the different models and techniques used, with a particular focus on the newly developed organoid models. We discuss how these models can be effectively utilized in microorganism-host interaction studies and address their associated limitations.

Tolerance of high hydrostatic pressure (HHP) is the hallmark of deep subsurface microorganisms, while its mechanisms remain under-investigated. This study explores HHP adaptation in the piezotolerant bacterium Orenia metallireducens across its near-full pressure range (0.1-40&#x2009;MPa). At inhibitory pressure (40&#x2009;MPa), the organism redirected carbon flux toward more favorable energy generation and biosynthesis using ferric mineral as the "electron sink." Furthermore, both universal and pressure-dependent strategies enabled the organism to withstand varying pressures. These findings highlight the role of iron minerals in microbial HHP adaptation and reveal novel survival strategies, advancing our understanding of deep-life evolution and biogeochemical impacts.

Flavonoids have significant medicinal potential; however, the reliance on plant extraction for their production poses a substantial barrier to their practical application. Innovative approaches like enzymatic cascade catalysis and microbial synthesis present promising avenues for the efficient production of these structurally complex compounds. In this study, two non-natural flavonoids (compounds 7 and 8) were synthesized, with compound 7 displaying antifungal properties. This was achieved through the cascade enzymatic catalysis of naringenin, a key intermediate in flavonoid biosynthesis, using glycosyl- and prenyltransferases. Additionally, metabolic engineering was used to bolster the supply of the dimethylallyl pyrophosphate precursor via the isopentenol utilization pathway in Escherichia coli, facilitating the de novo creation of non-natural flavonoids. Consequently, the titers of compounds 7 and 8 reached 47.4 and 6.6&#x2009;mg/l, respectively. This research highlights the potential of modular enzyme assembly in generating bioactive flavonoid derivatives and establishes a sustainable platform for the discovery of non-natural flavonoids.

Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation-Sequencing (fCUT&Tag-Seq) protocol specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae, Neurospora crassa, Fusarium graminearum, and Sporisorium scitamineum, showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.

The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage-host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.

Bacterial persisters show tolerance to bactericidal antibiotics and play essential roles in chronic infections; however, the general mechanisms underlying persister formation and antibiotic tolerance remain insufficiently characterized. In this study, the Escherichia coli Keio library was used to identify genes involved in ciprofloxacin tolerance by culturing each mutant to the late stationary phase (to induce persistence via starvation), followed by dilution into fresh medium for antibiotic exposure. This two-step, genome-wide screening approach enabled the identification of 37 ciprofloxacin-sensitive mutants with diverse biological functions and 11 ciprofloxacin-tolerant mutants related to amino acid and &#x3b2;-nicotinamide adenine dinucleotide (NAD&#x207a;) biosynthesis, with 25 genes being identified as persister-related genes for the first time. Notably, sensitive mutants (&#x394;atpC, &#x394;atpF, &#x394;ruvC, and &#x394;rnr) were specifically sensitive to quinolone antibiotics, whereas tolerant mutants (&#x394;metR, &#x394;leuB, and &#x394;nadB) showed tolerance to ampicillin and gentamicin. Importantly, adenosine triphosphate (ATP) levels were downregulated in ciprofloxacin-tolerant mutants and upregulated in ciprofloxacin-sensitive mutants, implying a negative correlation between ATP levels and ciprofloxacin tolerance among these genetically distinct persisters. This negative correlation was further observed when ATP levels in different mutants were chemically modulated using specific metabolites, nutrients, and respiration inhibitors. In addition, ciprofloxacin persistence across different mutants was found to correlate closely with antibiotic uptake and reactive oxygen species (ROS) levels. Collectively, these findings establish a universal role for ATP in the ciprofloxacin tolerance of genetically diverse persisters under varying resuscitation conditions, conceivably through the modulation of antibiotic uptake and ROS accumulation, and it is implied that the provision of abundant nutrients is potentially beneficial for anti-persister chemotherapy in clinic settings.

Helicobacter pylori is a major gastric pathogen with increasing antibiotic resistance, creating an urgent need for new therapeutic strategies. We screened 37 pure compounds and 9 herbal extracts for anti-H. pylori activity and identified berberrubine as the most potent agent, with a minimum inhibitory concentration of 11&#x2009;&#x3bc;g/ml. Berberrubine exhibited bacteriostatic effects by inducing oxidative stress and disrupting membrane integrity, as demonstrated by transcriptomic analysis, reactive oxygen species (ROS) accumulation, and structural damage, all of which were alleviated by the antioxidant N-acetylcysteine. Similar inhibitory effects were observed in Escherichia coli, indicating broader antimicrobial potential. This study provides the mechanistic evidence of berberrubine's activity against H. pylori, highlighting its promise as a candidate for development into alternative therapies to address antibiotic resistance.

Central carbon metabolism is thought to link reactive oxygen species (ROS) with antibiotic-mediated bacterial death. During enrichment screening of Escherichia coli with the first-generation quinolone oxolinic acid, unstable antibiotic-tolerant mutants containing deficiencies in purB were obtained. Examination of a stable deletion mutant of purA, a gene functionally related to purB, revealed reduced lethality of oxolinic acid and ciprofloxacin. This deletion mutation had little effect on the minimal inhibitory concentration (MIC) of quinolones, thereby demonstrating that the observed protection from killing was attributable to antibiotic tolerance. AMP synthesis was blocked by the &#x394;purA mutation, and ciprofloxacin tolerance was reversed by exogenous AMP supplementation. Because AMP is a precursor of ATP, interference with ATP synthesis occurs in the &#x394;purA mutant. RNA-Seq analysis showed that, prior to antibiotic stress, transcript levels of NADH:quinone oxidoreductase genes were reduced by the purA deficiency, thereby predisposing E. coli to antibiotic tolerance through reduced respiration. During ciprofloxacin exposure, the purA deficiency also suppressed the surge in expression of tricarboxylic acid (TCA) cycle and ATP synthesis genes, as well as the accumulation of intracellular ATP and ROS. Thus, wild-type PurA, and by extension the downstream enzyme PurB, directs AMP toward an antibiotic-mediated, ROS-dependent death pathway. Overall, defects in PurA/PurB-mediated adenosine ribonucleotides de novo biosynthesis reveal a novel quinolone tolerance mechanism that is initiated outside central carbon metabolism; tolerance is likely attributable to a limited supply of AMP, resulting in reduced ATP synthesis and suppression of ROS accumulation.

Cardiovascular diseases (CVDs) are the leading cause of mortality globally. Developing countries, including Pakistan, face a significant burden of CVD risk factors. Mobile health (mHealth) interventions have shown potential in promoting physical activity (PA) and reducing sedentary behavior; however, their use in CVD risk prevention, particularly among high-risk, urban sedentary employees remains underexplored. This study aims to develop and evaluate the feasibility and potential efficacy of a mobile-based Lifestyle Intervention for Employees (m-LIfE) to improve PA among sedentary bank employees in Karachi, Pakistan. A multiphase-sequential design will be conducted in two phases across branches of three commercial banks (one public and two private) in Karachi. In phase 1, a cross-sectional study will be conducted to estimate 10-year and lifetime risk for CVD events among bank employees, followed by focus groups discussions and interviews to explore employees' awareness, perceptions, and preferences regarding CVD prevention. These findings will inform the development of the m-LIfE app using a human-centered design (HCD) approach. In phase 2, a pilot cluster randomized controlled trial will be conducted to assess the feasibility (primary outcome) and potential efficacy of m-LIfE on PA (secondary outcome) over 12&#xa0;weeks, with a 4-week post-intervention follow-up. Bank branches will serve as clusters, with four randomized to m-LIfE and four to routine care. The m-LIfE app will deliver healthy lifestyle content for behavior change, and routine-care participants will receive paper-based educational pamphlets on CVD prevention. m-LIfE will use an HCD approach to co-create the intervention with participants, ensuring contextual relevance and addressing barriers faced by sedentary employees. This approach complements global evidence while accounting for unique cultural, organizational, and individual factors shaping lifestyle behaviors in Pakistan. This trial was registered with ClinicalTrials.gov on 20th May 2025 (Identifier: NCT06981247).

Dissimilatory sulfite reduction (DSR) has been essential to microbial energy metabolism in the biogeochemical sulfur cycle since the Paleoarchean Era. However, due to the lack of an integrated assessment of geological record and genomic data, the evolutionary origin of DSR remains elusive in terms of time, habitat, and genetic basis. In this study, we reconstructed the evolutionary pathways and the ancestral sequences of Dsr proteins by mining metagenomes ranging from mesothermal to hyperthermal environments. A phylogenetic analysis of the key catalytic enzyme, DsrAB, and other Dsr proteins indicates that the earliest and most basic functional cascade, DsrABCNM, emerged prior to the latest common ancestor of several basal branching DsrAB clusters encoded by bacteria and archaea. Using a molecular dating strategy that calibrates the protein tree with a species tree, we predicted that the DSR originated 3.508 billion years ago (Ga). This finding strongly confirms the earliest geological evidence of DSR (&#x2009;~&#x2009;3.47 Ga). Further predictions from ancestral sequence reconstruction indicate that the optimal catalytic temperature of DsrA at the time of DSR origin was approximately 73&#xb0;C, which is consistent with the petrographic and geochemical evidence in early Archean hydrothermal deposits. After its hot origin, DsrA diversified into subclades that adapted to various temperature levels following the Great Oxidation Event. This is exemplified by the evolution of the reductive archaeal-type DsrA. Our results synchronize the molecular ages with the geological record, which advances our understanding of the earliest DSR systems and highlights the enzymatic adaptations of microbial life in the Archean biosphere.