Biology and Life Sciences

Abstract: Avian Metapneumovirus (aMPV) is an emerging viral pathogen causing many outbreaks in chickens all over the world. There are several subtypes of the MPV circulating in chickens in the US. The current circulating subtypes of the virus in the US chicken are (A, B, and C). Despite the availability of some aMPV vaccines in the US, most of these vaccines are based on foreign strains, particularly European aMPV-A/B strains. Their protective efficacy against contemporary U.S. aMPV isolates has not been fully established. The main goal of this study is to integrate the most recent aMPV genome sequencing data and the machine learning tools to design a novel aMPV. The machine learning tools such as epitope mapping, molecular docking, and immune simulation were used to design the multiepitope DNA vaccine based on the top ranked epitopes of two major surface proteins of the virus (F and G). The top ranked seventeen epitopes representing B cells, CD4 and CD8 epitopes were linked using linkers, with IL-18 added as an adjuvant. The selected epitopes showed high antigenicity, no toxicity and no allergenicity values among the screened epitopes. The molecular docking analysis of the designed vaccine construct showed a high binding affinity to the MHC class I and II epitopes to chicken alleles. The immune simulation analysis of this vaccine construct showed the potential to induce robust immune response including the humoral and the cell mediated immunity. Further functional studies are required to test the immunogenicity and the efficacy of this novel vaccine before applications using chickens and turkey.

Abstract: Nucleosomes integrate diverse histone variants to organize chromatin and coordinate genome maintenance. In plants, the structural interplay between replication-associated histones and DNA damage -responsive histone variants remain poorly understood. Here, we determine cryogenic electron microscopy structures of an Arabidopsis thaliana nucleosome containing the replication-associated histones H2B.6 and H3.1 together with the DNA damage-responsive variant H2AXa.The mononucleosome structure, resolved at 2.73 Å preserves the canonical nucleosome architecture. Unexpectedly, the reconstituted mononucleosomes formed defined higher-order assemblies in the absence of linker DNA or chemical crosslinking. Two distinct nucleosome-nucleosome arrangements, parallel and right offset, were identified. The right offset closely resembles the human counterpart, whereas the parallel arrangement exhibits slight displacement of H2B structural elements at the inter-nucleosomal interface. Together these structures reveal how a distinct histone composition preserves canonical nucleosome architecture while supporting multiple defined modes of nucleosome packing, providing structural insights into conserved and divergent features of H2AX containing chromatin organization across plants and animals.

Abstract: In July 2026, an outbreak of a cattle illness characterized by high fever, diarrhoea and reduced milk production, often affecting whole herds, was detected across several regions of northern Switzerland. To investigate the cause of the outbreak a broad range of diagnostic methods was applied, including pathogen specific RT-qPCRs, ELISA, serum neutralization test, virus isolation and metagenomic next generation sequencing. A Shamonda virus (SHAV) was detected in samples from affected cattle, followed by successful virus isolation and complete genome sequencing of all three segments. Viral RNA was also detected in tissue samples from aborted calves, including brain and spleen, supporting the need for further investigation of the virus in the context of reproductive disease. These findings document the first detection and molecular characterization of a SHAV in Switzerland and demonstrate its widespread circulation in cattle during the 2026 outbreak.

Abstract: Background: dengue fever is a viral infection caused by the Dengue Virus (DENV), a positive-sense RNA virus belonging to Flaviviridae family. The virus is transmitted to humans through the bite of the Aedes aegypti mosquito. Worldwide, between 100 and 400 million cases are reported annually. The infection in humans can cause three clinical forms which are related to severity: dengue without warnings signs, dengue with warnings signs, and severe dengue. Mutations in some genes that code for immune system proteins have been linked to the appearance of severe clinical forms of the disease, including TNF-α. The TNF-α plays a key role in the immunopathogenesis of the disease: mutations in this gene can increase the production of TNF-α, favoring cytokine storms and vascular damage, which is associated with severe clinical forms of the disease. Therefore, it is important to study the immunopathogenesis of the disease in the human to try to predict and control the onset of severe clinical forms and fatal cases. Methods: This study explores the link between TNF-α promoter gene polymorphism and dengue severity. We re-cruited 92 laboratory-confirmed dengue cases, including 30 dengue without warnings signs, 43 dengue with warnings signs, and 19 severe dengue. Genotyping was done using amplification by PCR (polymerase chain reaction), Sanger sequencing, and bio-informatics analysis of the TNF-α gene promoter mutations. A logistic regression assessed the association between -308 G/A TNF-α mutation and dengue severity. Results: the mutated genotype -308 G/A TNF-α was detected in 10.5% of severe cases. Interestingly, Severe cases showed higher odds of carrying the mutation than those without warnings signs. Severe cases were twice as likely to possess this mutation compared to those with warnings signs, however, no statistically significant differences were found. Genotype frequencies were consistent with Hardy-Weinberg equilibrium (p = 0.112). The estimated post hoc statistical power was 11.3%. Conclusions: Although no statistically significant association was detected, the TNF-α -308G/A polymorphism showed a trend toward increased susceptibility to severe dengue, with a higher frequency among severe cases than among patients with or without warnings signs.

Abstract: Hypermobile Ehlers–Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD) are clinically diagnosed connective tissue conditions without validated molecular biomarkers. We investigated whether miRNA-mediated post-transcriptional regulation converges between them. miRNome sequencing was performed in dermal fibroblasts from 12 hEDS, 12 HSD, and 24 control individuals, followed by qPCR assessment of 25 selected differentially expressed miRNAs (DE-miRNAs) in the discovery samples and an independent pooled fibroblast cohort, and integration with a previously generated transcriptome. No miRNA reached statistical significance in the direct hEDS-HSD comparison, whereas pooled hEDS/HSD analysis identified 88 DE-miRNAs versus controls (42 upregulated and 46 downregulated). Experimentally supported targets of the upregulated and downregulated miRNAs overlapped, in the expected inverse direction, with 388 downregulated and 27 upregulated genes, respectively. These candidate regulatory axes converged on TGF-β/BMP, Wnt/Hippo, focal-adhesion and cytoskeletal programs, and on a more focused chemokine-inflammatory module. The results define a shared fibroblast miRNA signature across hEDS and HSD, nominate miRNA-mRNA axes for mechanistic testing, and provide a rationale for future validation in individual patients, disease-relevant tissues, and circulating biofluids.

Abstract: Metabolic endpoint assays enable rapid screening but provide limited information on treatment kinetics, heterogeneity, and recovery. Here, we established a multistep workflow combining MTS-based metabolic screening with electric cell–substrate impedance sensing (ECIS), a label-free cell-based biosensor, to functionally prioritize complex plant extracts in head and neck squamous cell carcinoma (HNSCC) models. Seventeen soluble extracts were screened at 50 µg/mL for 24 h in four HNSCC cell lines and human adipose-derived stem cells. Selected candidates were subsequently characterized by real-time impedance monitoring, multifrequency analysis, and model-derived barrier resistance. Extract 16 showed the most favorable metabolic selectivity profile. ECIS resolved no growth-inhibitory effects, sustained impedance suppression, heterogeneous responses, and transient suppression followed by partial recovery. Cross-model analysis identified distinct barrier-resistance dynamics, particularly in CAL-33 and Detroit 562. Comparison with the PI3Kα inhibitor Inavolisib revealed partially overlapping but non-identical metabolic response profiles and no consistent enhancement by combination treatment. Extract 16 was further associated with junctional redistribution, F-actin remodeling, and PARP processing, whereas reproducible caspase 3/7 activation was not detected. Thus, integrating metabolic endpoint screening with multiparametric ECIS monitoring provides greater functional resolution and supports the prioritization of complex bioactive samples for subsequent chemical and mechanistic investigation.

Abstract:

Helicobacter pylori (H. pylori) and Streptococcus gallolyticus subspecies gallolyticus (SGG) have been implicated in colorectal carcinogenesis, but whether host immune responses to these bacteria influence colorectal cancer (CRC) survival remains unclear. We investigated whether pre-diagnostic antibody responses to H. pylori and SGG antigens were associated with overall and CRC-specific mortality within a prospective cohort study. We included 471 incident CRC cases from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Pre-diagnostic antibody responses to 13 H. pylori and 11 SGG antigens were measured using multiplex serology. Cox proportional hazards models estimated hazard ratios (HRs) and 95% confidence intervals (CIs), adjusted for key confounders. Overall H. pylori seropositivity was defined as ≥4 positive antigens, while SGG seropositivity was defined using a 6-marker panel (≥2 positive markers). Over a median follow-up of 9.2 years, 254 all-cause and 188 CRC-specific deaths were observed. Overall seropositivity to H. pylori and SGG, including the SGG 6-marker panel, was not associated with CRC-specific or overall mortality. However, seropositivity to H. pylori catalase was associated with reduced CRC-specific mortality (HR=0.71, 95% CI: 0.49–1.01). Pre-diagnostic antibody responses to specific H. pylori antigens, particularly catalase, may be associated with improved CRC survival, while overall seropositivity to H. pylori and SGG showed no consistent associations.

Abstract: Secretion of recombinant proteins from microbial cell factories is a promising strategy for recombinant protein biomanufacturing as it simplifies downstream processing. In this study, we sought to intensify the Lactococcus lactis high-cell-density culture, using basic fibroblast growth factor 2 (FGF2) as a model recombinant protein. We implemented a perfusion strategy using tangential flow filtration for cell retention, allowing continuous removal of inhibitory metabolites while replenishing fresh nutrients. When conventional 2×GM17 medium was used, the approach outperformed batch cultivation, achieving a 5.2-fold increase in biomass and a 2.6-fold increase in secreted FGF2, reaching a titer of 9000 µg·L⁻¹. Concurrently, we developed a bioprocess model for L. lactis grown in a fortified spent cell culture medium, enabling systematic exploration of operating conditions. A Pareto front was generated for FGF2 titer against media usage, and perfusion profiles balancing both competing objectives were identified. An experimentally selected operating point validated the model predictions, yielding a final OD₆₀₀ of 49.2 and FGF2 titer of 2166 µg·L⁻¹ FGF2, which were 8-fold and 5-fold higher than batch process respectively. Overall, this work demonstrates a perfusion-based intensification strategy for L. lactis and highlights the utility of model-guided process decision making to enhance productivity while reducing waste.

Abstract: Abiotic stresses such as drought, salinity, temperature extremes, flooding, and exposure to toxic elements restrict crop productivity by disrupting plant water relations, hormonal regulation, redox homeostasis, osmotic balance, ion transport, photosynthesis, and reproductive development. Plant-associated microorganisms offer a biologically based means of supporting crop acclimation, although their effectiveness depends on the microbial strain, host genotype, environmental conditions, and capacity to colonise and persist in the target system. This review critically examines the physiological, biochemical, and molecular mechanisms through which beneficial bacteria and fungi influence plant responses to abiotic stress. Particular attention is given to microbial modulation of abscisic acid, auxins, cytokinins, ethylene and 1-aminocyclopropane-1-carboxylate metabolism, together with less extensively characterised interactions involving gibberellins, jasmonates, salicylic acid, brassinosteroids, and strigolactones. Microbial regulation of reactive oxygen and nitrogen species, enzymatic and non-enzymatic antioxidant systems, compatible-solute metabolism, Na⁺/K⁺ homeostasis, root hydraulic conductivity, aquaporins, and rhizosphere hydraulic properties is also evaluated. The evidence indicates that these mechanisms operate as interconnected regulatory networks rather than as independent protective processes. Particular caution is required when interpreting changes in stress markers, hormone concentrations, antioxidant activities, osmolytes, or transporter transcripts because these responses may indicate either enhanced acclimation or differences in stress severity. Stronger mechanistic evidence is provided by microbial biosynthetic mutants, complemented strains, hormone- or signalling-impaired plants, direct hydraulic measurements, protein localisation, ion-flux analysis, and isotopic tracing. Future progress will require time-resolved and tissue-specific studies that integrate microbial colonisation with functional plant measurements, followed by validation across representative soils, genotypes, climates, and management systems. Microbial inoculants should therefore be regarded as context-dependent components of integrated crop-management strategies rather than universal substitutes for good agronomic practices.

Abstract: Neurodegenerative diseases (NDs) comprise a heterogeneous group of disorders characterized by complex etiologies, multifactorial pathogenic mechanisms, and remarkable clinical and molecular variability. Their increasing prevalence poses an important challenge to healthcare systems, society and global economy. Despite decades of research, effective disease-modifying therapies remain elusive, highlighting the urgent need for reliable disease models capable of unraveling pathogenic mechanisms and supporting the development of personalized therapeutic strategies. Modeling NDs remains inherently challenging, since disease onset and progression arise from the interplay among genetic factors, environmental exposures, aging, and individual pathological conditions. Consequently, similar insults may lead to distinct molecular trajectories and clinical outcomes in different subjects. Reproducing this heterogeneity, together with the prolonged temporal evolution that characterizes NDs over decades in humans, represents a major limitation of current experimental models. Here we provide a comprehensive overview of the modeling strategies currently available for the study of NDs, spanning conventional two-dimensional cellular systems, advanced patient-derived three-dimensional organoids, complementary in vivo animal models, and rapidly evolving in silico approaches. For each model category, we discuss its historical development, current applications, future perspectives, and, critically, principal strengths and limitations in reproducing disease biology and supporting translational research. We emphasize that the growing synergy between in vitro, in vivo, and in silico methodologies, empowered by multimodal data integration and artificial intelligence, is expected to accelerate biomarker discovery, improve disease modeling, reduce experimental burden, and ultimately foster the development of personalized disease-modifying therapies for NDs.

Abstract: Cladophialophora is a melanized fungal genus that includes environmental, transitional, and clinically relevant lineages, yet its diversity in tropical montane soils remains poorly understood. In this study, ITS2 metabarcoding was used to investigate Cladophialophora diversity in five soil samples collected along an altitudinal gradient (0-3015 m a.s.l) in the Sierra de las Minas Biosphere Reserve, Guatemala. From a total of 2,848 fungal ASVs obtained from soil samples, 38 were assigned to Cladophialophora and analyzed through UNITE-based taxonomic assignment, maximum-likelihood phylogenetic reconstruction, lineage classification, abundance profiling, and exploratory multivariate analyses. The recovered ASVs clustered predominantly within an environmental clade, whereas transitional, basal, and clinically affiliated clades were represented by fewer ASVs and lower relative abundances. Eleven phylogenetically defined lineages were recognized, with the C. floridana lineage being the most widespread and abundant, followed by the transitional C. lanosa lineage. ASVs affiliated with the C. bantiana clinical lineage were detected at low abundance and were restricted to a warmer zone of Zacapa. Overall, these findings provide the first metabarcoding-based and phylogenetically informed characterization of Cladophialophora in Guatemala and Sierra de las Minas soils and establish a baseline for future ecological and taxonomic studies.

Abstract: DNA methylation (DNAm) signatures may capture molecular variation associated with cognitive phenotypes, but saliva-based scores for subjective memory are poorly characterized. We developed MMS-32, a frozen 32-CpG salivary DNAm score derived from an independent EPIC-array comparison of individuals reporting Very bad memory (n = 1,117) versus Very good memory (n = 2,400). Candidate loci were screened using Mann-Whitney U tests; MMS-32 comprises all candidate CpGs with discovery p < 1 × 10⁻⁴ and uses signed discovery mean differences as normalized weights. The frozen score was evaluated in an independent male dataset (n = 1,044). Among single-category memory responses, MMS-32 discriminated Very bad (n = 16) from Very good (n = 66) memory with AUC = 0.861 (95% bootstrap CI 0.736–0.957; p = 8.50 × 10⁻⁶). A broader Bad/Very bad versus Good/Very good contrast yielded AUC = 0.603 (p = 0.0079). Linear dependence on chronological age was negligible (R2 = 0.0022; Pearson r = −0.047, p = 0.133), and the extreme-memory association remained significant after age adjustment. MMS-32 is therefore a research-stage, memory-associated methylation signature rather than a biological-age score. Independent validation against objective (diagnosed dementia, MRI etc.) and longitudinal cognitive outcomes is required.

Abstract: Crohn’s disease (CD) is a multifactorial inflammatory bowel disease (IBD) arising from interactions among genetic susceptibility, immune dysregulation, intestinal microbiota, and environmental factors. However, genetic and environmental determinants of IBD in Djibouti remain poorly characterized. This study aimed to characterize the demographic and clinical profile of IBD and investigate the distribution and association of major CARD15/NOD2 variants with CD in a Djiboutian population. A total of 150 participants aged 18–30 years were enrolled, including 50 CD patients, 50 ulcerative colitis (UC) patients, and 50 healthy controls. Demographic, clinical, familial, lifestyle, and environmental characteristics were recorded. Genomic DNA isolated from intestinal mucosal biopsies was analyzed by PCR followed by Sanger sequencing for R702W, G908R, and 1007fs variants. Carrier and allele frequencies were assessed using odds ratios (ORs), 95% confidence intervals (CIs), and categorical statistical tests. CD patients showed higher carrier frequencies of R702W (24.0%), G908R (16.0%), and 1007fs (20.0%) than controls (10.0%, 6.0%, and 8.0%, respectively). Allele-level analysis indicated increased odds for R702W (OR=2.55), G908R (OR=2.67), and 1007fs (OR=2.59), although associations were not statistically significant. Abdominal pain and chronic diarrhoea predominated clinically, with greater weight loss in CD and rectal bleeding in UC. Notably, all CD participants reported khat consumption before disease onset. These findings suggest potential genetic susceptibility to CD and identify khat consumption as a hypothesis-generating environmental factor requiring further investigation.

Abstract: The increasing specialization of crop and livestock production has disrupted traditional nutrient cycling in agricultural systems, creating a dual challenge of excessive dependence on synthetic fertilizers and inefficient utilization of livestock manure. Crop–livestock circular agriculture offers a promising pathway to address these interconnected problems by recoupling animal production, manure management, and crop cultivation within an integrated nutrient-recycling framework. This review critically synthesizes recent advances in crop–livestock circular agriculture, with particular emphasis on manure valorization, nutrient recovery and reuse, enabling treatment technologies, crop–livestock nutrient matching, and regionally adapted implementation models. Current evidence demonstrates that appropriately managed manure recycling can partially substitute synthetic fertilizers, improve soil fertility and structure, enhance nutrient-use efficiency, and reduce nutrient losses and associated environmental pressures. Technologies including solid–liquid separation, aerobic composting, anaerobic digestion, and emerging resource-recovery approaches further expand the potential for converting livestock waste into fertilizers, energy, and other value-added agricultural inputs. However, the environmental and agronomic benefits of these systems depend strongly on balancing manure-derived nutrient supply with crop demand and local land carrying capacity. This requirement is particularly important in the hilly agricultural regions of Southwest China, where fragmented farmland, dispersed livestock production, complex terrain, and high transportation costs constrain the direct adoption of large-scale centralized models. Locally adapted strategies integrating decentralized manure treatment, nearby land application, and coordinated regional nutrient allocation may therefore provide more practical solutions. Despite substantial progress, broader implementation remains limited by spatial mismatches between manure production and cropland demand, insufficient technological adaptation, economic constraints, and a lack of long-term system-level assessments. Future research should move beyond individual waste-treatment technologies toward integrated crop–livestock management that combines nutrient budgeting, precision manure application, resource recovery, digital decision support, and region-specific governance. Such advances are essential for transforming livestock manure from an environmental liability into a strategic nutrient resource and for accelerating the transition toward resource-efficient, low-impact, and resilient agricultural systems.

Abstract: Introduction: Cantú syndrome, or hypertrichotic osteochondrodysplasia, is a rare genetic disorder characterized by generalized hypertrichosis, macrosomia, cardiomegaly, and distinctive coarse facial features. What is unique about this case is the clinical challenge of distinguishing syndromic hypertrichosis from premature pubarche in early childhood, emphasizing the necessity for multidisciplinary and targeted endocrine monitoring in these patients and contributing to the sparse literature on its long-term management. Main concerns and clinical findings: An 8-year-old girl with Cantú syndrome presented for a growth assessment and evaluation of early body hair development. She exhibited striking generalized hypertrichosis on her legs, back, and face, alongside a complex cardiovascular history and chronic kidney disease. Primary diagnoses, interventions, and outcomes: A thorough physical and endocrinological evaluation revealed normal baseline androgens and elevated SHBG, effectively differentiating her syndromic hypertrichosis from a reversible endocrine imbalance (e.g., premature adrenarche or pubarche). Interventions have been largely observational for her hypertrichosis due to the lack of targeted treatments and complicating comorbidities, while her cardiovascular and renal conditions continue to be managed multidisciplinarily. Conclusion: Cantú syndrome requires a highly individualized approach. Clinicians must remain vigilant for evolving endocrine dysfunctions, particularly regarding growth and pubertal development, while supporting the profound psychosocial burden caused by the currently untreatable hypertrichosis.

Abstract: This study evaluated deficit irrigation (DI; 55% of crop evapotranspiration (ETc) from BBCH 76 to four days after harvest, followed by rewatering) compared with full irriga-tion (FI; 100% ETc) in ‘Regina’ trees. DI reduced soil water, tree water status, light-saturated photosynthesis (ASAT) and stomatal conductance (gs). However, temporal changes in ASAT were weakly related with leaf water potential (ΨL) and closely related to gs and mesophyll conductance (gm). DI modified non-structural carbon partitioning in leaves, increasing sorbitol concentration by 27% and decreasing sucrose by 57% without affecting total soluble sugars. At the same time, lignin fluorescence decreased under DI whereas leaf mass per area (LMA) and stomatal density remained unaffected. After harvest and rewatering, ΨL increased in both treatments, but leaf gas exchange activity dropped accompanied by increases in lignin fluorescence and LMA. Despite an 8% de-crease in fruit size, fruit yield was maintained, improving water productivity by 67%. Our results indicate that sweet cherry responses to DI are not solely determined by water status, but also involve carbon partitioning, leaf structural traits and source-sink dy-namics, allowing yield to be maintained at the expense of fruit size.

Abstract: Islatravir is a novel nucleoside reverse transcriptase translocation inhibitor under clinical investigation for the treatment of HIV-1 infection. While we previously documented high-level phenotypic resistance to islatravir (ISL) in HIV-1 subtype C variants harboring combinations of type 2 thymidine analogue mutations (TAM2) along with the M184V resistance-associated mutation, the primary contributor to the observed phenotype remained unclear. This report dissects the relative contribution of M184V to ISL resistance in the context of TAM2-containing variants through systematic reversion analysis. We show that reverting mutant 184V to wildtype M184 resulted in substantial sensitization to ISL, with fold-change reductions ranging from 8.1 to 13.4-fold across three TAM2 genetic backgrounds. Contrary to the well-documented antagonism between M184V and TAMs in zidovudine resistance, these two resistance pathways exhibit cooperative rather than antagonistic interactions in the context of ISL resistance. These findings provide critical evidence for refinement of genotypic resistance interpretation algorithms for ISL and have direct implications for the future clinical management of ISL-based antiretroviral therapy in people living with HIV. Importantly, elucidating the mechanistic basis of M184V-TAM2 cooperation would establish a more predictive framework for understanding how complex resistance mutation patterns impact ISL susceptibility and guide treatment decisions in virologically experienced individuals.

Abstract:

Getah virus (GETV) is a mosquito borne Old World alphavirus that has recently increased its geographic range, particularly in China. GETV causes rheumatic disease in horses, but in recent years its primary impact has been lethal infections in newborn piglets. Herein we characterize a lethal Ifnar1-/- mouse model of GETV, which recapitulated many of the features seen in piglets. These include lethality, high viremia, diarrhoea, and splenomegaly, with histopathological findings aligning with those seen in piglets and indicating severe, acute, multi-organ inflammatory immunopathology. RNA-Seq of spleens from GETV infected Ifnar1-/- mice and subsequent bioinformatic analyses illustrated a cytokine storm response that included robust type I interferon signatures and a pattern of cytokine/chemokine signatures indicative of viral sepsis/septic shock. Ifnar1-/- mice thus offer a framework for understanding, and a mouse model for, GETV disease in piglets. The model also indicates that lethal GETV infection in piglets ay involve viral sepsis/septic shock.

Abstract: Background/Objective Krüppel-like factor 5 (KLF5) as a member of the zinc finger protein family has been reported in hepatocellular carcinoma (HCC). However, the KLF5 as a novel biomarker for patients with chronic liver diseases (CLD) remains to be identified. This study investigated the clinical values of KLF5 in CLD malignant transformation. Methods KLF5 transcripts from the TCGA database were analyzed with functions and related signaling pathways. Under the ethics committee consent, microarrays were constructed from HCC and noncancerous tissues. KLF5 distribution and expression were analyzed by multiplex immunofluorescence or Western blotting. Bloods were collected from a cohort of cases with CLD. Serum KLF5 levels were quantitatively detected by an enzyme-linked immune-sorbent assay. Results KLF5 at mRNA levels were signifi-cantly upregulating expressed (P<0.001) in HCC tissues more than these in normal livers from the TCGA database. Strong KLF5 expressions were verified in human HCC cell lines or tissues. Clini-copathological features of high KLF5 levels were remarked related (P<0.001) to tumor size, AFP level, HBV infection, tumor/node/metastasis stage and overall survival rate. Furthermore, the in-cidence of KLF5 in the HCC group were significantly higher (P<0.001) more than those in cases with liver cirrhosis or chronic hepatitis. Also, KLF5 was an independent prognostic factor for HCC. Interestingly, the co-expressions of KLF5 with Wnt3a promoted CLD malignant transfor-mation. Conclusion Upregulated KLF5 was associated with CLD malignancy and could be as a promising diagnostic or prognostic biomarker for HCC.

Abstract: Future International Space Station (ISS) mini-gardens must provide fresh produce repeatedly while maintaining water and nutrient conditions within a small, reusable root-zone volume. This study evaluated the operational performance of staggered planting and selective harvesting over four months in a ground-based Utah Reusable Root Module (URRM) simulator operated as a zero-discharge system. Five independent root modules (RMs), providing a total planted area of approximately 0.40 m², were planted with leafy greens, bonsai basil, and dwarf tomato. The system produced 4,299 g of fresh edible biomass, with produce available on 77 of the 100 days within the harvest period. Daily water use remained below approximately 2 L d⁻¹ for most of the trial, and peak demand occurred at different times among RMs. RMs containing established tomato plants behind newly planted leafy greens showed lower water-use peaks and biomass production, suggesting incomplete use of module capacity. Lettuce performed well during the first cycle but was less consistent in subsequent cycles, whereas mizuna and kale provided more consistent production under reused-media conditions. Bulk electrical conductivity (bulk EC) declined during periods of rapid crop growth, indicating that nutrient depletion, rather than salinity accumulation, was the main root-zone risk under zero-discharge operation. These results indicate that ISS mini-garden operations can distribute harvests and water demand over time, but crop calendars should coordinate planting dates, crop architecture, harvest timing, residue management, reused-media constraints, and root-zone nutrient monitoring across independently managed modules.