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Recombinant protein production in Escherichia coli is frequently limited by aggregation into inclusion bodies, reducing the recovery of active protein. In this study, we compared two naturally occurring Arabidopsis thaliana PRLIP1 variants from the Columbia-0 and Wassilewskija accessions, which differ by 13 amino acids, during temperature-controlled heterologous expression as GFP fusion proteins. GFP fluorescence, protein distribution between soluble and insoluble fractions, and esterase activity recovered after alkaline solubilization and refolding were analysed. Reduced cultivation temperatures increased GFP fluorescence and recovered esterase activity, although most of the recombinant protein still remained associated with inclusion bodies. Differences between the two PRLIP1 variants were observed in fluorescence intensity, inclusion body accumulation, and recovered esterase activity, indicating that natural PRLIP1 variation is associated with altered recombinant protein behaviour. Sequence-based hydropathy analysis revealed no major global differences in overall hydrophobicity, whereas aggregation prediction indicated subtle local differences in aggregation-prone regions. These results suggest that naturally occurring allelic variation can provide a useful first-pass approach to identify sequence-associated differences in folding-related behaviour and functional recovery, while residue-level causal mechanisms require further mutational analysis.

Strain evolution poses a global threat, yet medical resources often fall short of meeting the demands of such public health emergencies. To examine how constrained medical resources and random factors influence disease transmission dynamics, this study incorporates the limited number of hospital beds into a stochastic infectious disease model that accounts for strain evolution and transmission rate affected by the logarithmic Ornstein-Uhlenbeck process. The primary contributions include deriving sufficient conditions that guarantee the existence, uniqueness and boundedness of the positive global solution for the stochastic model, identifying thresholds governing disease extinction and persistence and establishing sufficient conditions for the existence of a stationary distribution, based on which we compute the probability density function of the model to quantify the final size of the disease from a statistical perspective. Numerical simulations indicate that: (i) when strain 1 exhibits dominant transmissibility, it secures a dominant competitive position via its transmission advantage, while strain 2 sustains endemic transmission by exploiting recovered individuals, enabling the long-term coexistence of both strains. By contrast, when the two strains have comparable transmissibility, strain 1, which depends exclusively on susceptible individuals for transmission and survival, is eliminated via competitive exclusion by strain 2 (which can infect individuals recovered from strain 1), and is ultimately driven to extinction; (ii) for epidemic prevention and control, we should not only constrain the mean transmission rate of the disease below the epidemic threshold, but also reserve an adequate control safety margin against stochastic fluctuations in transmission rate; (iii) in the early stage of an epidemic when medical resources are scarce, it is necessary to rapidly expand hospital bed capacity. Once resource supply matches the epidemic demand, the focus of prevention and control should be shifted to optimizing the efficiency of resource scheduling. These results have certain significance for preventing and controlling diseases with such transmission patterns.

Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains.IMPORTANCEHighly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.

Many ecosystems worldwide are experiencing chronic anthropogenic nutrient enrichment, which often increases plant productivity while reducing species richness. Although nutrient inputs are now declining in some regions, the potential benefits of this reduction depend on the reversibility of enrichment impacts. In turn, ecosystem recovery can be determined by the enrichment history, that is, the rate and duration of nutrient enrichment. Here, we quantify how nutrient enrichment history shapes community recovery dynamics using a four-decade grassland experiment that examines the joint effects of nutrient enrichment rate and duration with: (1) three durations of nutrient enrichment and recovery: one decade of enrichment followed by three decades of recovery, three decades of enrichment followed by one decade of recovery, or continuous enrichment for four decades; and (2) nutrient enrichment at a gradient of rates ranging from atmospheric deposition to agricultural fertilization. Our results showed nutrient enrichment increased plant biomass and reduced species richness, with higher nutrient addition rates leading to more rapid and sustained species loss and biomass increase, even over short enrichment periods. We assessed recovery dynamics following cessation as increases in species richness and declines in community biomass relative to control conditions, because of the tight coupling between richness and biomass in many communities. We found that the reversibility of enrichment effects depended on enrichment duration, with prolonged enrichment slowing recovery of both species richness and biomass, especially at high enrichment rates. However, biomass recovered more rapidly than species richness following cessation. These findings highlight that recovery trajectories of biodiversity and ecosystem functioning depend jointly on enrichment rate and duration, underscoring the need for restoration strategies that account for nutrient legacies and their determinants. 全球范围内许多生态系统正在经历由长期人类活动引起的营养物质富集，这往往会提高植物群落生产力，同时降低物种丰富度。尽管部分地区的营养物质输入正在减少，但这种减少能否带来生态效益，取决于营养物质富集效应的可逆性。而生态系统恢复的过程又可能受到营养物质富集历史的影响，即营养物质添加的速率与持续时间。本研究基于一项持续四十余年的草地实验，量化了不同营养物质富集历史如何影响植物群落恢复动态。该实验同时控制了养分添加速率与持续时间，包括:(1)三种不同的养分添加与恢复历史:添加10年后停止并恢复32年、添加32年后停止并恢复10年，以及持续添加42年；(2)八种养分添加速率，覆盖从大气氮沉降到农业施肥水平(0–272 kg N·ha⁻¹·yr⁻¹)。 研究结果表明，即使在较短的富集时间内，较高的养分添加速率也会导致更快速且持续的物种丧失与生物量增加。由于许多植物群落中物种丰富度与生物量之间存在紧密耦合关系，养分添加停止后，我们通过物种丰富度以及群落生物量相对于对照条件的差距来衡量群落恢复动态。研究发现，营养物质富集效应的可逆性受到养分添加持续时间的显著影响；长期养分添加会延迟物种丰富度与群落生物量的恢复，尤其是在高养分添加速率下更为明显。不过，在停止养分添加后，群落生物量的恢复速度显著快于物种丰富度。这些结果表明，生物多样性与生态系统功能的恢复轨迹受到养分添加速率与持续时间的共同影响，凸显了充分考虑营养物质遗留效应及其决定因素对于生态恢复策略制定的重要性。.

Paraquat poisoning is one of the most lethal toxicological emergencies, particularly in low- and middle-income countries. Despite several proposed treatment regimens, no single approach has conclusively demonstrated a reduction in mortality. Early clinical manifestations may be nonspecific, and prompt recognition in the emergency department is critical. We report the unexpected survival of a young woman who ingested a potentially fatal dose of paraquat, with emphasis on long‑term radiological follow‑up and dynamic hematological changes. A 22‑year‑old Iranian woman presented to the emergency department approximately 48 h after reportedly ingesting 20 mL of a 20% paraquat solution in a suicide attempt. She had initially been evaluated at another facility shortly after ingestion but had refused admission against medical advice. She developed oropharyngeal mucosal ulcerations, odynophagia, hemoptysis, and acute kidney injury (peak serum creatinine 3.9 mg/dL). Laboratory data revealed a triphasic white blood cell pattern, including profound leukopenia followed by a marked rebound after granulocyte colony‑stimulating factor (G‑CSF) administration. A chest CT obtained at admission showed no pulmonary involvement; however, a follow-up CT performed on Day 14 demonstrated diffuse bilateral ground‑glass opacities and patchy consolidations consistent with acute alveolar injury. The patient received early multidisciplinary management including high‑dose corticosteroids, cyclophosphamide, antioxidant therapy, broad‑spectrum antibiotics, antimicrobial prophylaxis against opportunistic infections, nutritional support, and electrolyte correction. Clinical improvement was observed within 72 h, and she recovered without requiring dialysis or mechanical ventilation. A six‑month follow‑up chest CT showed complete radiological resolution with no evidence of pulmonary fibrosis. This case suggests that complete pulmonary and renal recovery may be achievable in clinically severe paraquat poisoning. The six‑month imaging follow‑up documents the complete resolution of acute lung injury, while the dynamic leukocyte pattern illustrates the severity of bone marrow suppression and subsequent recovery. These findings highlight the potential value of early recognition, intensive multidisciplinary management, and structured follow‑up, particularly in resource‑limited settings.

We isolated 61 actinomycete strains from diverse animal-derived samples. Although Streptomyces predominates in soil environments, actinomycetes recovered from animal samples were primarily non-Streptomyces taxa, including rare genera such as Micromonospora sp. The antimicrobial activities of the isolated actinomycetes were evaluated, and several strains exhibited antibacterial and antifungal activities. These findings highlight animal-derived actinomycetes as promising sources of bioactive natural products.

2,4-Diacetylphloroglucinol (DAPG) is a valuable antimicrobial compound with significant agricultural potential, suffers bioproduction limitations from host toxicity and inefficient downstream processing. This study engineered DAPG-hyper-tolerant Escherichia coli via adaptive laboratory evolution (ALE) starting from a phloroglucinol-tolerant strain. Optimized shake-flask fermentation of evolved Bdt03 yielded 330.52 mg/L DAPG (17.84-fold of the wild type), and the yield from the whole fermentation broth could be further increased to 391.36 mg/L. A novel organic solvent-free extraction method recovered DAPG from fermentation broth with over 98% yield via acidification, cold incubation and centrifugation, simplifying downstream processing. Genomic resequencing identified several key mutations underlying DAPG tolerance, which were validated and stacked to precisely construct strain Bb03 with enhanced production and tolerance. This work addresses the critical bottlenecks in DAPG biosynthesis by enhancing host tolerance and developing a sustainable downstream processing strategy, and also offers valuable genetic insights for constructing high-yield DAPG-producing strains and advancing the application of DAPG-responsive genetic circuits in synthetic biology.

Concurrent pyomyositis and septic arthritis without systemic symptoms is uncommon and may delay diagnosis. A 36-year-old immunocompetent male presented with isolated right knee swelling, without fever or trauma. Magnetic resonance imaging revealed joint effusion and myositis in surrounding muscles. Joint aspiration confirmed Methicillin-resistant Staphylococcus aureus septic arthritis. Despite intravenous antibiotics, a growing intramuscular abscess in the gastrocnemius was detected on serial ultrasound. Surgical drainage was performed. The patient was treated with IV vancomycin for 2 weeks, followed by oral linezolid, and recovered fully. Pyomyositis should be considered in septic arthritis with atypical presentations. Serial imaging and inflammatory markers are essential for guiding treatment.

Non-typhoidal Salmonella (NTS) is a leading bacterial pathogen responsible for foodborne outbreaks worldwide, with poultry products being a major source of human salmonellosis. While previous studies have predominantly focused on Salmonella transmission in the chicken production chain, ducks have received comparatively little attention as a potential reservoir host. In this study, we investigated Salmonella contamination across a commercial duck breeder-hatchery-grow-out (BHG) production system in Inner Mongolia, Hebei, and Shandong, China, comprising five breeder duck farms, one hatchery, and one commercial grow-out duck farm. From January 2022 to July 2025, a total of 7765 samples were collected across the duck BHG production system, from which 796 Salmonella isolates were recovered, yielding an overall prevalence of 10.25%. Serotyping analysis identified 16 serovars, dominated by S. Typhimurium (52.51%, 418/796), followed by S. Anatum (18.72%, 149/796) and S. Enteritidis (16.21%, 129/796). To further explore genomic relationships and potential transmission routes, 212 of the 418 S. Typhimurium isolates, representing diverse sampling time points, production stages, sample sources, locations, and antimicrobial-resistance profiles, were selected for whole-genome sequencing (WGS). Core-genome single nucleotide polymorphism (cgSNP) analysis revealed the presence of both vertical and horizontal transmission of S. Typhimurium from breeder ducks to commercial ducks. Antimicrobial susceptibility testing showed that 83 S. Typhimurium isolates (26.35%) exhibited antimicrobial resistance (AMR) to more than three classes of antibiotics. These isolates exhibited high resistance rates to ampicillin, amoxicillin, tetracycline, nalidixic acid, and streptomycin. A comparative genomic analysis of multidrug resistance genes identified an IS26-mediated chromosomal replacement event, leading to the replacement of a chromosomal region with a multidrug resistance region (MRR). This genomic rearrangement may contribute to the persistence and dissemination of multidrug-resistant clones within the production system. Overall, these findings demonstrate that Salmonella circulating in breeder ducks and farm environments can disseminate through eggs and the hatchery to commercial meat ducks, suggesting that the hatchery may serve as key points for amplification and cross-contamination. These results underscore the need for strengthened hatchery biosecurity, including routine surveillance, strict workflow separation, and enhanced cleaning and disinfection, to reduce the dissemination of antimicrobial-resistant Salmonella along the duck production chain.

Liquid and glass are two closely related disordered states of matter. Whether each can exhibit two or more structurally distinct yet chemically identical forms have been debated for decades and the relationship between polymorphism in equilibrium liquids and that in non-equilibrium glasses remains elusive. Here, we address these long-standing issues in two prototype glass-forming metallic alloys using in situ high-pressure high-temperature synchrotron X-ray diffraction in diamond anvil cells. We identify distinct liquids above their melting temperatures and corresponding glasses formed upon melt quenching at different pressures, as evidenced by reproducible discontinuous changes in multiple diffraction-derived structural descriptors. Comprehensive structural and compositional characterizations consistently demonstrate that the recovered samples are fully amorphous and chemically homogeneous glasses. By continuously monitoring the formation and transformation pathways with in situ structural probes, our study provides compelling experimental evidence for the more general existence of liquid and glass polymorphism in densely packed metallic alloy systems.

The production of the biopolymer poly-3-hydroxybutyrate (P3HB) by the halophile Halomonas boliviensis in fed-batch cultivations using bench-scale bioreactors was re-examined aiming to improve productivity and decrease production costs. To improve productivity, controlled feeding of glucose and nitrogen source, and supplementation of extra monosodium glutamate (MSG), phosphate, and trace elements were assessed. Non-aseptic conditions were investigated as a strategy to reduce costs related to medium and equipment sterilization. Further, to reduce production costs, glucose-rich hydrolysates derived from industrial residues of the red macroalga Gelidium corneum were used as an alternative carbon source to glucose. These hydrolysates were produced through hydrothermal pretreatment to remove residual agar followed by enzymatic hydrolysis. Enzyme recycling was evaluated as a strategy to improve process economics. After hydrolysis, the enzymatic cocktail was recovered using an ultrafiltration membrane and reused in the next batch. Supplementation of trace elements and increased phosphate addition contributed to a significant increase of cell dry weight (CDW), P3HB titer and overall P3HB volumetric productivity up to 103.1 ± 1.9 g/L, 70.9 ± 0.3 g/L, and 0.92 ± 0.04 g/L·h, respectively. Under non-aseptic conditions, P3HB titers and productivities were comparable to those achieved under axenic conditions. Algal hydrolysates proved to be an effective glucose source, yielding results similar to those obtained with commercial glucose. This study shows that H. boliviensis is a promising platform for P3HB production and can efficiently convert carbohydrate-rich residual biomass under non-sterile conditions.

Recycled cellulose fibers recovered from paper industry waste were valorized as sustainable reinforcement materials for the development of boric acid/silica-modified cementitious composites with balanced mechanical and thermal performance. A Taguchi-TOPSIS multi-response optimization approach was employed to systematically evaluate the combined effects of cellulose fiber, boric acid, and silica contents on compressive strength, heat capacity, thermal conductivity, and water absorption. Structural characterization by FT-IR, XRD, and SEM-EDX confirmed that the principal cement hydration phases were preserved after modification, while silica-rich formulations exhibited a relatively denser and more homogeneous matrix morphology. Thermogravimetric analysis showed that cellulose incorporation increased the total mass loss from approximately 12-14% for the reference cement to 17-20% for the modified composites, whereas boric acid and silica partially moderated this effect by maintaining residual mass values of 80-85% at elevated temperatures. Among the experimentally evaluated formulations, the Taguchi-TOPSIS multi-response optimization identified the A2B2C3 composition as the optimum formulation, achieving a compressive strength of 38.73 MPa, corresponding to a 52.63% improvement over the reference mixture, together with a 37.06% increase in heat capacity, while simultaneously reducing thermal conductivity and water absorption. These results demonstrate that the combined incorporation of recycled cellulose fibers, boric acid, and silica provides an effective strategy for developing sustainable cementitious composites with improved multifunctional performance while promoting the high-value utilization of paper industry waste.

The exponential growth of biomedical literature creates a cognitive bottleneck in drug target discovery, particularly for identifying therapeutically relevant mechanisms beyond established pathways. In ocular neovascularization, anti-VEGF therapies are standard of care, yet non-response and resistance remain critical unmet needs. We present an integrated computational framework combining Graph Retrieval-Augmented Generation (GraphRAG)-based literature mining, pathway co-localization analysis, and deep learning-based druggability assessment for systematic target prioritization. Using 5562 angiogenesis-related PubMed abstracts, we constructed a vascular knowledge graph (17 842 nodes; 9555 edges) and applied pathway co-localization with vascular endothelial growth factor A (VEGF-A) as a biological filter. As a validation step, the workflow recovered four targets-fibroblast growth factor 2, transforming growth factor-beta 1, interleukin-1 beta, and matrix metalloproteinase-9-already supported by clinical or advanced preclinical development, demonstrating concordance with expert-driven selection. Iterative querying subsequently identified two additional mechanistically supported candidates, fibroblast growth factor 1 and hepatocyte growth factor, sharing receptor tyrosine kinase-centered pathways with VEGF-A but lacking clinical evaluation in ocular neovascularization. Deep learning-based structural analysis (DeepSite and PocketMiner) identified high-confidence ligandable pockets for all six candidates. This work demonstrates how GraphRAG can systematically mine existing literature to recover known targets and surface literature-supported candidates that may be underprioritized for translational development. Rather than claiming de novo discovery, we emphasize the framework's utility as a scalable, transparent, and reproducible methodology for overcoming citation bias and literature overload. The workflow is generalizable to other complex, literature-rich disease domains.

Melanoma is one of the most aggressive forms of cancer in human due to its ability to invade tissues and metastasize. The aim of this work is to examine the effect of our patented compound Prunus spinosa Trigno + Nutraceutical Activator Complex (PsT + NAC®) on primary (WM115), metastatic (WM266-4), and malignant (A375) human melanoma cell lines. The data show that PsT + NAC® induced a dose- and time-dependent reduction in cell viability in all melanoma cell lines, particularly in the metastatic WM266-4. Persistent morphological changes indicative of cell death were observed, which remained irreversible even after the cells recovered from treatment. The treatment with PsT + NAC® altered cell migration and motility by remodeling of actin cytoskeleton. Cell cycle analysis revealed a G2/M phase arrest in the primary WM115 cells, and a G1 phase arrest-at lower concentrations-in the metastatic WM266-4 cells, and in the malignant A375 cells. As the treatment concentration increased, all melanoma cell lines showed an increase in the sub-G1 population, associated with apoptosis. Western blotting analysis revealed that lower concentrations of PsT + NAC® induced a protective autophagic response, while higher concentrations triggered caspase-dependent apoptosis. These results demonstrate the efficacy of PsT + NAC® in inhibiting the growth of BRAF-mutant melanoma cells.

Vibrio parahaemolyticus is traditionally associated with raw seafood, but cross-contamination in commercial catering can create overlooked hazards. We investigated a gastroenteritis outbreak affecting 88 individuals to determine how a non-seafood vehicle became the route of transmission. We conducted integrated epidemiological and environmental traceback investigations. Whole-genome sequencing (WGS) and core-genome multilocus sequence typing (cgMLST) were applied to analyze the genetic relatedness and resistome of the recovered isolates. Traceback strongly implicated rice noodles as the most likely vehicle. The outbreak was traced to a prerequisite program failure (raw clam exudate dripping onto uncovered noodles) followed by a critical control point failure (insufficient blanching for 1-2 min). WGS and cgMLST supported these findings: clinical and rice-noodle isolates were genetically identical (0-1 SNPs, tdh+), whereas a V. parahaemolyticus isolate from cooked bean sprouts was phylogenetically distant (>1300 allelic differences) and tdh-negative, excluding it as the cause. The outbreak clone also exhibited a multidrug-resistant (MDR) phenotype, including Colistin resistance and the CARB-22 β-lactamase gene. These results indicate that cross-contaminated food matrices can act as reservoirs for clinically relevant resistance determinants. This underscores the need for strict physical barriers, validated thermal processing, and high-resolution genomic surveillance to prevent opportunistic pathogen transmission in commercial catering.

Kosakonia radicincitans is primarily recognized as a plant-associated and environmentally adapted member of Enterobacteriaceae, whereas recovery from human clinical specimens remains uncommon. Routine biochemical identification may misassign recently reclassified Kosakonia species to closely related Enterobacter taxa. We characterized strain ZJG61129, a K. radicincitans isolate recovered from bile during endoscopic retrograde cholangiopancreatography (ERCP) in a patient with choledocholithiasis. Colony morphology, VITEK 2 Compact identification, MALDI-TOF MS identification and antimicrobial susceptibility testing were performed. Whole-genome sequencing, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), 16S rRNA and core-genome phylogeny analyses, whole-genome comparison, pan-genome analysis, and resistance/virulence-associated gene screening were used for taxonomic confirmation and genomic characterization. ZJG61129 formed smooth, moist colonies on blood agar and MacConkey agar. VITEK 2 Compact assigned the isolate to the Enterobacter cloacae complex with 92% confidence, whereas MALDI-TOF MS identified it as K. radicincitans with a score of 2.259. The genome consisted of a single circular chromosome of 5,510,622 bp with a GC content of approximately 54.0%. ANI and dDDH analyses confirmed assignment to K. radicincitans, and 16S rRNA together with core-genome phylogeny placed ZJG61129 within the Kosakonia lineage. Comparative genomics showed a conserved genomic backbone, substantial genomic plasticity, and no clear source-associated genomic structuring in the current dataset. ZJG61129 was susceptible to all antimicrobial agents tested. In silico resistance and virulence gene analyses did not indicate high-risk acquired resistance or a clearly high-virulence genotype. ZJG61129 represents a human biliary-derived, environmental-like K. radicincitans isolate that may be misassigned by routine biochemical identification. Genome-based analysis is valuable for accurate recognition of uncommon, recently reclassified Enterobacteriaceae from biliary specimens.

Proteolytic microbial consortia are key drivers of protein hydrolysis in complex organic substrates. In anaerobic digestion systems, such as those used for biogas production from sewage sludge, this process constitutes the initial and rate-limiting step. Despite their importance, proteolytic microorganisms remain poorly characterized due to the complexity of environmental microbiomes and the limitations of conventional cultivation and screening methods. Here, we present a label-free microfluidic protocol for the high-throughput cultivation and characterization of proteolytic microorganisms. Single microbial cells are encapsulated in gelatin droplets and grown clonally, where proteolytic activity is detected through image-based analysis of droplet shape changes. Enrichment of individual proteolytic cultures is achieved using a separate microfluidic device that enables passive droplet sorting. Taxonomic characterization of sorted droplets by 16S rRNA gene sequencing revealed a fivefold higher number of amplicon sequence variants (ASVs), and a more diverse array of proteolytic strains were recovered compared with conventional skim milk agar screening (SMA). Taken together, this microfluidic workflow allows accurate and fast enrichment of proteolytic strains. Our approach advances the understanding of proteolytic communities in sewage sludge and opens new opportunities for targeted microbial recovery in waste-to-energy applications. Proteolytic microorganisms drive the initial and rate-limiting step of protein degradation in anaerobic digestion systems, such as sewage sludge biogas production, yet their diversity and function remain poorly characterized due to the limitations of conventional cultivation methods. We present a label-free droplet microfluidic workflow that enables high-throughput, single-cell cultivation, functional screening, and selective enrichment of proteolytic microbes directly from complex communities. This approach substantially improves the recovery and diversity of proteolytic strains compared with traditional assays, providing a powerful tool to study hydrolytic consortia and to enhance microbial discovery for waste-to-energy and other biotechnological applications.

Ice-ice disease (IID) is widely associated with environmental stress and microbial presence in seaweed farms, but its expression under ex situ conditions remains poorly understood. This study examined the associated culturable bacterial community in independently maintained K. alvarezii stock cultures classified as healthy or diseased based on characteristic symptoms. Bacteria were isolated from healthy tissue, diseased tissue, and their corresponding bulk waters, identified using 16S rRNA gene sequencing, and analyzed through phylogenetic reconstruction, species richness estimation, and community similarity metrics. A total of 31 bacterial isolates representing six unique taxa were recovered. Diseased tissue exhibited higher bacterial richness and a distinct tissue-associated bacterial composition compared with healthy tissue. Healthy tissue exclusively harbored Rossellomorea aquimaris, whereas diseased tissue contained Vibrio and Photobacterium species, with representative isolates showing agarolytic and carrageenanolytic activity. In contrast, bulk waters from healthy and diseased cultures contained Pseudoalteromonas species. Although the study focuses on microbial associations, the emergence of IID under controlled gene bank conditions highlights the potential interaction of host condition, culture environment, routine handling, and opportunistic bacteria. Together, these factors illustrate the "black box" dynamics of IID in ex situ conditions and provide insights for disease monitoring and management in seaweed gene banks.

Herein, we report a novel synthesis of the polymer-supported Brønsted-acidic ionic liquidPS-[(SO3H)4C4Im]-[OTf]as an efficient and recyclable heterogeneous catalyst for the Groebke-Blackburn-Bienaymé (GBB) multicomponent reaction. The catalyst was synthesized from Merrifield resin (polystyrene) and fully characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). Its catalytic activity was then evaluated in GBB model reactions, with the optimal conditions determined using 50 mg mmol-1 of PS-[(SO3H)4C4Im]-[OTf] in ethanol as solvent under microwave heating at 150 °C for 1 h. A diverse library of imidazo-fused heterocycles (e.g., imidazo-[1,2-a]-pyridines, imidazo-[2,1-b]-thiazoles, and benzo-[d]-imidazo-[2,1-b]-thiazoles) was synthesized using the optimized conditions and the corresponding products were obtained in moderate to excellent yields (34-91%), depending on the starting aminoazole. Furthermore, the heterogeneous catalyst could be easily recovered by filtration after each reaction cycle and reused for up to six consecutive cycles with no significant loss of integrity as well as catalytic activity (average yield 86 ± 3.5%). These results demonstrated the potential of this polymer-supported Brønsted-acidic ionic liquid as a sustainable catalyst for acid-catalyzed multicomponent reactions applied to the synthesis of nitrogen-based heterocycles of interest in Medicinal Chemistry.