Abstract: Background: The human microbiome, particularly the gut microbiota, is essential for health maintenance and disease prevention. Probiotics, defined as live microorganisms that provide health benefits to the host, are increasingly recognized for their capacity to inhibit pathogenic bacteria through competitive exclusion. This meta‑analysis systematically evaluates the effectiveness of probiotics in reducing pathogen colonization within the human gut. Methods: A comprehensive literature search was conducted across PubMed and Scopus from October 2018 to August 2023, identifying in vivo studies reporting competitive exclusion by probiotics. Thirty studies met the qualitative criteria, with four contributing quantitative data. Results: The pooled odds ratio for the impact of probiotics on pathogen exclusion was 1.68 [1.13–2.51], demonstrating a statistically significant benefit (p < 0.01). Heterogeneity was minimal (I² = 0%), supporting the robustness of the findings. Conclusions: Results underscored the efficacy of Lacticaseibacillus, Limosilactobacillus, and Bifidobacterium strains in competitive exclusion. These findings support the integration of probiotics into therapeutic strategies for managing gastrointestinal infections and highlight the need for future research on strain‑specific effects and optimal dosing.

Abstract: Background: Fungal symbionts are critical for host plant survival but are vulnerable to abiotic stresses such as low temperature, which limits their agricultural utility. Armillaria mellea is an essential fungal partner for the cultivation of the valuable medicinal orchid Gastrodia elata (G. elata). Enhancing its cold tolerance is a key step toward stabilizing G. elata production. Methods: Based on G. elata transcriptome data, a manganese superoxide dismutase gene (GeSOD7) was identified and heterologously expressed in Escherichia coli for enzymatic characterization. The gene was then overexpressed in A. mellea via Agrobacterium tumefaciens-mediated transformation. Transgenic and wild-type strains were subjected to cold stress (13 °C for 45 days), after which physiological, biochemical, and molecular responses were analyzed. Results: Recombinant GeSOD7 showed optimal activity at pH 6.0 and 60 °C, with inhibition under high concentrations of metal ions, especially Mn2+ and Cu2+. Overexpression of GeSOD7 in A. mellea significantly improved hyphal growth and fresh weight under cold stress. Transgenic strains exhibited higher activities of catalase and glutathione peroxidase, increased accumulation of glutathione and proline, and reduced levels of hydrogen peroxide and malondialdehyde. Expression of genes involved in glutathione synthesis and peroxide detoxification was coordinately upregulated. Conclusions: This study demonstrates that heterologous expression of a plant-derived Mn-SOD can effectively enhance the cold stress tolerance of a symbiotic fungus by boosting its enzymatic and non-enzymatic antioxidant systems. These findings provide a novel genetic strategy for improving stress resilience in agriculturally important fungi and contribute to the sustainable cultivation of G. elata.

Abstract: Ex vivo functional testing for multiple myeloma is rapidly evolving, yet no single assay has reached the level of reliability and clinical utility needed for routine decision-making. Existing approaches generally fall into three categories comprising 2D cultures, 3D models, and dynamic systems. Each contributes valuable but incomplete insight into therapeutic response. Among these, 2D assays remain the most mature, with the most extensive clinical correlations to date, though their simplified architecture limits their ability to reflect the full complexity of the bone marrow microenvironment. 3D systems, including spheroids and matrix-based organoids, offer improved preservation of tumor heterogeneity and microenvironmental cues. These platforms show emerging clinical relevance and may hold advantages over traditional 2D formats, and validation efforts are developing. Dynamic systems including microfluidic models and perfused bone-marrow mimetics represent the most physiologically ambitious category, yet their technical intricacy and early stage of development have so far limited broad clinical correlation.Altogether, the current landscape highlights substantial progress but lacks an optimal assay. In this review, we take the unique approach of examining published ex vivo tests that have demonstrated a level of clinical correlation. We evaluate their respective formats, strengths and limitations, and discuss considerations for what an ideal future assay may encompass.

Abstract: Background: Hepatitis B virus (HBV) is the smallest partially double-stranded, reverse-transcribing DNA virus, with four open reading frames (ORFs) encoding viral proteins. It is classified into nine geographically distributed genotypes (A–I). In Kenya, the molecular characterization of HBV among patients seeking medical care remains poorly defined. Objectives: This observational study aimed to characterize HBV among patients seeking medical care in the endemic region of Kenya, focusing on circulating genotypes and ORF mutations. Methodology: Serum samples were collected from the outpatient departments of selected health facilities, with demographic and clinical information extracted from patients’ medical records. Hepatitis B surface antigen (HBsAg) was tested at the facilities, and a total of 85 HBsAg-positive samples were collected for molecular analysis. The basal Core promoter and pre-core (BCP/PC), polymerase, and surface regions of the viral genome were amplified and sequenced to determine genotypes and to profile their mutations. Results: Out of 85 HBsAg-positive samples, 38 samples tested positive for HBV DNA, and 26 samples were successfully sequenced. HBV genotype A was prevalent 73.1% (19/26), followed by genotype D 23.1% (6/26), and genotype E 3.8% (1/26). Genotype A sequences clustered with both A1 Asian and African subgenotypes, whereas genotype D clustered with subgenotypes D6 and D1. All HBV genotype A, D, and E sequences were serotypes adw2, ayw2, and ayw4, respectively. HBV core promoter mutations (A1762T/G1764A) were detected in both genotype D and genotype A isolates. The pre-core G1896A mutation was highly prevalent in genotype D samples (5/6; 83.3%) but was not observed in genotypes A or E. Analysis of mutations within the aa determinant region revealed genotype-specific patterns: genotype A predominantly harbored V14A, P46H, S58C, and P67Q substitutions; genotype E showed N59S; and genotype D exhibited V14A, C69stop, S104T, and W182stop mutations. Two drug resistance mutations (V191I and A194T) were present in two chronic patients, one with genotype A and the other with genotype D. Conclusion: HBV genotypes A and D are the most prevalent among Kenyan patients with chronic HBV infection. The presence of point mutations in the ORFs among patients seeking medical care highlights the need for a molecular surveillance to better understand the viral diversity and its potential clinical and public health implications.

Abstract:

Astragalus membranaceus (AM) is a traditional medicinal and edible herb with well-documented immunomodulatory activities; however, its application in functional beverages is limited by the low bioavailability of its bioactive constituents. Probiotic fermentation has emerged as an effective strategy to enhance the nutritional and functional properties of herbal materials, yet the underlying metabolic mechanisms remain insufficiently understood. In this study, untargeted metabolomics based on ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry (UHPLC-Orbitrap MS) was employed to comprehensively characterize metabolic alterations in AM aqueous extracts before and after fermentation with Pediococcus acidilactici (P. acidilactici) for 48 h. Multivariate statistical analyses combined with pathway enrichment analysis were used to identify differential metabolites and key metabolic pathways affected by fermentation. A total of 659 significantly altered metabolites were identified, including 350 upregulated and 309 downregulated metabolites after fermentation. These metabolites were mainly associated with organic acids, flavonoids, amino acid derivatives, nucleotides, and phenylpropanoids. Notably, fermentation markedly enhanced metabolites related to arginine biosynthesis, carbon metabolism, and nicotinate and nicotinamide metabolism, accompanied by a substantial accumulation of functional compounds such as lactate, phenyllactic acid, indolelactic acid, and nicotinamide adenine dinucleotide (NAD⁺). Overall, P. acidilactici fermentation induced extensive metabolic reprogramming of AM aqueous extracts, leading to the enrichment of multiple bioactive metabolites and the activation of key functional processes. These findings provide mechanistic insights into probiotic fermentation of medicinal and edible herbs and offer a scientific basis for the development of value-added fermented AM beverages with improved nutritional and functional properties.

Abstract: Central nervous system (CNS) disorders constitute a significant global health challenge; however, the development of therapeutic agents is considerably impeded by the difficulty in delivering effective concentrations within the brain. This comprehensive review delineates the current landscape of computational modeling techniques employed to address the formidable challenges associated with CNS drug delivery, with a particular emphasis on the anatomical barriers and physiological transport mechanisms pertinent to major neurological diseases. We categorize modeling approaches ranging from the atomistic scale, including molecular dynamics simulations of drug-blood-brain barrier (BBB) interactions, to macroscopic continuum and Physiologically Based Pharmacokinetic (PBPK) models that elucidate systemic distribution and overall brain exposure. We critically assess these models concerning established delivery routes, such as intranasal and intrathecal administration, as well as emerging methods, including focused ultrasound-mediated BBB opening and targeted nanoparticle delivery. This review underscores the growing importance of integrating complex physiological phenomena, such as glymphatic flow and cerebrospinal fluid (CSF) dynamics, into predictive models. Finally, we explore the emerging opportunities involving multiscale digital twins of the CNS that integrate molecular interactions, vascular hemodynamics, CSF and perivascular flow, and parenchymal transport within patient-specific anatomical geometries. The role of machine learning and surrogate modeling in expediting the prediction of drug transport parameters and optimizing delivery strategies is also examined. By providing a structured overview of current computational tools, this review aims to guide researchers in the design of more robust computational platforms for CNS drug delivery.

Abstract:

Melanin accumulation is the primary cause of skin hyperpigmentation, and most existing cosmetic agents address this process by inhibiting melanogenesis. In contrast, strategies that directly decolorize or degrade melanin remain largely unexplored. In this study, we report a novel biobased cosmetic ingredient derived from onion (Allium cepa)–associated endophytic fungi that exhibits direct melanin decolorization alongside skin-whitening and anti-aging activities. Endophytic fungi were isolated from onion tissues, and aqueous extracts were prepared to ensure cosmetic-grade compatibility. Preliminary screening demonstrated exceptional melanin-reducing capacity among the isolates, with a maximum reduction of 97.83%, highlighting their strong melanin degrading potential. A selected isolate, identified as Aspergillus brasiliensis (ACL05), was further investigated to elucidate the influence of sterilization methods on bioactivity. The autoclaved culture filtrate retained substantial melanin-reducing activity (62.85%), whereas ultrasonication-based cell inactivation resulted in significantly lower activity (32.54%), indicating that heat-stable extracellular metabolites are primarily responsible for melanin decolorization. A cosmetic essence formulated using the sterile ACL05 extract achieved a measurable melanin reduction of 15.39%, demonstrating formulation feasibility and functional efficacy. Beyond melanin decolorization, the ACL05 extract exhibited multifunctional anti-aging properties, including inhibitory activities against tyrosinase, collagenase, and elastase, as well as significant antioxidant capacity as determined by the DPPH assay. Collectively, these findings reveal, for the first time, the potential of onion-derived endophytic Aspergillus brasiliensis as a sustainable source of multifunctional cosmetic bioactives. This work introduces a new paradigm for skin-whitening based on direct melanin decolorization while simultaneously addressing skin aging, supporting the development of next-generation biobased cosmetic ingredients.

Abstract: Background: MicroRNAs (miRNAs) are an important class of non-coding RNAs that regulate gene expression by binding to target mRNAs and influencing cellular processes such as differentiation, proliferation, and apoptosis. Dysregulation in miRNA expression has been reported to be implicated in many human diseases, including cancer, cardiovascular, and neurodegenerative disorders. Identifying disease-related miRNAs is therefore essential for understanding disease mechanisms and supporting biomarker discovery, but time and costs of experimental validation are the main limitations. Methods: We present a graph-based learning framework that models the complex relationships between miRNAs, diseases, and related biological entities within a heterogeneous network. The model employs a message-passing neural architecture to learn structured embeddings from multiple node and edge types, integrating biological priors from curated resources. This network representation enables the inference of novel miRNA–disease associations, even in sparsely annotated regions of the network. The approach was trained and validated on a dataset benchmark using ten replicated experiments to ensure robustness. Results: The method achieved an average AUC–ROC of ~98%, outperforming previously reported computational approaches on the same dataset. Moreover, predictions were consistent across validation folds and robustness analyses were conducted to evaluate stability and highlight the most important information. Conclusions: Integrating heterogeneous biological information and representing them through graph neural representation learning offers a powerful and generalizable way to predict relevant associations, including miRNA–disease, and provide a robust computational framework to support biomedical discovery and translational research.

Abstract: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder marked by abdominal discomfort, altered bowel habits, and comorbidities like anxiety, fatigue, and poor sleep. Existing treatments often fall short of long-term relief and fail to address root causes, leaving many patients with persistent symptoms. This contributes to a substantial burden on individuals and healthcare systems, highlighting the need for new, mechanism-based therapies. Emerging evidence suggests that gastrointestinal symptoms may be caused by the cumulative burden of epithelial injury and insufficient time for mucosal maintenance and repair. This paper explores the hypothesis that structured intermittent fasting regimes could represent a safe, low-cost, and underutilized therapeutic strategy for IBS by promoting gut renewal and restoring homeostasis. We review the multifactorial pathophysiology of IBS and explore how fasting may counter these mechanisms. Evidence from IBS and IBD studies shows that intermittent fasting can reduce inflammation, enhance autophagy, regulate gut motility, and reshape the microbiota, thereby strengthening the gut barrier and dampening immune responses. Notably, fasting induces autophagy, a key cellular recycling process essential for intestinal barrier maintenance and microbial defense, which may be impaired in IBS. Although clinical studies on fasting regimes and IBS are limited, evidence from related populations and mechanistic research supports further exploration. As a practical, circadian-aligned approach that does not restrict specific foods, intermittent fasting may reduce epithelial injury and allow time for repair. The shift from “what to eat” to “when to eat” offers a new, physiology-based potential tool for IBS treatment.

Abstract: Impairments in balance control are common across various clinical conditions and aging, necessitating reliable methods for assessment. This study introduces a novel, low-cost posturographic system based on an unstable spring-supported platform that calculates center of pressure (COP) displacement using angular measurements in two horizontal axes. A heterogeneous sample of 105 participants underwent repeated trials on both the novel system and a traditional firm platform under eyes-open and eyes-closed conditions. COP velocity was recorded and analyzed for reliability using intraclass correlation coefficients (ICC). Results showed significantly higher COP ve-locity on the unstable platform when visual input was removed, indicating greater re-liance on visual control under unstable conditions. The novel system demonstrated comparable reliability to traditional platforms, with ICC values exceeding 0.90 when mean values from three trials were used. No learning effect was observed on the un-stable platform, unlike the firm one. These findings suggest that the new system is a valid alternative for balance assessment, particularly effective in differentiating indi-viduals with varying balance capabilities under eyes-closed conditions. Its affordabil-ity and methodological soundness make it suitable for clinical use and broader screen-ing applications aimed at fall prevention.

Abstract: Poly-γ-glutamic acid (γ-PGA) is an important biopolymer produced by various species of Bacillus. Novel γ-PGA producers have shown strain-dependent nutritional and culture requirement that must be characterised and optimised to improve γ-PGA yields. The optimal nutritional and cultural condition for maximum γ-PGA titre in a newly identified γ-PGA producing strain Bacillus licheniformis DPC6338 was determined using one factor at a time (OFAT) and design of experiments (DOE). The optimal nutritional and culture condition for maximum γ–PGA titre in B. licheniformis DPC6338 was 67g/L glutamic acid, 32g/L tryptone, 75g/L glucose, 5g/L citric acid, 2g/L K₂HPO₄, 0.5g/L MgSO₄·7H₂O, 0.02 g/L FeCl₂·4H₂O, 0.1g/L CaCl₂·2H₂O, 0.5 g/L MnSO₄·H₂O, 2g/L ZnSO₄·7H₂O, 42°C, pH 6.5 – 7.0, 1% inoculum, at 250 rpm. Under optimised conditions in shake flask, maximum γ–PGA titre 75.35 ± 0.38 was obtained after 96h while peak productivity of 1.3 g/L/h occurred at 48 h, representing a 27% and 4% improvement in titre and productivity compared to the screening medium. Scale-up to bioreactor conditions significantly enhanced the final titre γ-PGA and early-phase volumetric productivity by ~30% and ~80% respectively. The results obtained in this study highlight the potential of B. licheniformis DPC6338 for industrial γ-PGA producing strain.

Abstract:

Mycophenolic acid (MPA), a secondary metabolite derived from fungal strains, is a therapeutic agent drawing significant attention due to its potential applications in organ transplant rejection, autoimmune disorders, and cancer cell inhibition. It also exhibits potent antiviral, antifungal, and antibacterial properties, positioning it as a candidate for next-generation antibiotics. Research is presently focused on bioprospecting for MPA-producing fungal strains with a broad activity spectrum to enhance clinical efficacy. In this study, 304 fungal strains were isolated from diverse marine sediments in central and southern Vietnam. Thin-layer chromatography (TLC) identified 25 strains capable of synthesizing MPA. Based on morphological characteristics, these were classified into three genera—Penicillium, Aspergillus, and Cladosporium—alongside two unidentified strains. Notably, high-performance liquid chromatography (HPLC) confirmed that strain MBLC9-138 possesses high MPA-producing potential, reaching 463.25 to 632.03 mg/L after 5–7 days of cultivation. Internal transcribed spacer (ITS) sequencing identified this strain as Cladosporium sp. MBLC9-138, marking the first report of MPA biosynthesis within this genus. Furthermore, MPA extracted from this strain exhibited significant antimicrobial activity against Escherichia coli (Gram-negative), Staphylococcus aureus, and Bacillus cereus (Gram-positive), with MIC values of 32, 64, and 16 µg/mL, respectively. These results highlight a promising bioactive candidate that could offer dual therapeutic benefits while potentially minimizing gastrointestinal side effects and antibiotic resistance. Simultaneously, Vietnamese marine sediments continue to be a rich source of material for isolating bioactive microorganisms, particularly MPA-producing strains.

Abstract: Banana bract mosaic virus (BBrMV) is the most economically damaging and deleterious Potyvirus pathogen (family, Potyviridae) of banana (2n = 3x = 33) that causes significant losses to banana production Asia. The BBrMV has a single-stranded, positive sense ssRNA genome of 9708 nucleotides. RNA interference (RNAi) is an evolutionarily conserved biological potent intracellular response mechanism in eukaryotic organisms and is an antagonist of virus replication. The current study focuses on the role of banana genome-encoded microRNAs (mac-miRNAs) targeting +ssRNA genome of the BBrMV using in-silico predictive algorithms, RNA22, RNAhybrid, TAPIR and psRNATarget. Mature banana locus-derived mac-miRNA sequences (n = 32) were tested for alignment with the BBrMV genomic +ssRNA (NCBI accession No. MG758140). In total we extrapolated 32 mature banana miRNAs, only two of which are potentially efficient miRNAs (mac-miR157b and mbg-miR397a) to have high-affinity target sites in the BBrMV genome. To identify emerging therapeutic targets, we utilized Circos software to develop a network illustrating potential mechanistic RNA-RNA interactions, based on robust prediction algorithms. Our findings provide the first in silico evidence of multiple dynamic tenacious interaction between banana high-confidence miRNAs and Potyvirus genome. This work represents a critical step towards proactive biosecurity preparedness, offering a predictive framework for engineering BBrMV-resistant banana plants and safeguarding domestic banana production.

Abstract: As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve an Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI) (asymptomatic plants), as well as in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in wheat blast hotspot district, Meherpur in Bangladesh. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as a modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.

Abstract: Background: microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression post-transcriptionally and are now recognized as central players in tumorigenesis, progression, and therapeutic resistance. Their remarkable stability in body fluids and close integration with oncogenic and tumor-suppressive pathways have positioned miRNAs as promising diagnostic, prognostic, and therapeutic biomarkers across diverse cancers. However, the dual and context-dependent nature of many miRNAs, together with methodological heterogeneity, complicates their translation into routine clinical practice.Methods: A comprehensive literature review was conducted using PubMed/MEDLINE, Embase, and Web of Science from 1993 to June 2025 to identify peer-reviewed studies evaluating miRNAs in human cancers. Articles were included if they investigated miRNAs in relation to cancer biology, biomarker potential (diagnostic, prognostic, or predictive), metastasis and EMT, the tumor microenvironment, or therapeutic modulation. Mechanistic, translational, and clinical studies were all considered. Data were extracted on cancer type, sample source, miRNAs studied, assay methods, clinical associations, and functional validation. Due to heterogeneity in design and reporting, a narrative thematic synthesis was undertaken rather than meta-analysis.Results: Evidence across multiple malignancies shows that specific miRNAs function as oncomiRs, promoting proliferation, survival, angiogenesis, immune evasion, and metastasis (for example, miR-21, miR-155, miR-10b, and the miR-17-92 cluster), while others act as tumor-suppressor miRNAs, constraining oncogenic signalling, cell-cycle progression, and metastatic spread (for example, miR-34a, miR-126, miR-145, miR-124, miR-15a/16-1, and the miR-200 family). Distinct expression signatures have been associated with early-stage disease, advanced stage, and metastatic patterns in solid tumors and hematological malignancies. Circulating and exosomal miRNAs show high stability and have demonstrated potential for non-invasive cancer detection, risk stratification, and monitoring of treatment response. Several miRNAs correlate with survival outcomes and resistance to chemotherapy, targeted agents, and immunotherapies. Preclinical models support miRNA-based therapies, including mimics to restore tumor-suppressor miRNAs and inhibitors (antagomirs) to silence oncomiRs, with emerging delivery platforms such as nanoparticles and engineered exosomes.Conclusion: microRNAs (miRNAs) emerge as integrative cancer biomarkers that reflect underlying genetic, epigenetic, and microenvironmental complexity. Current evidence indicates their potential to improve early detection, refine prognostic stratification, and inform therapeutic decision-making. However, heterogeneity in analytical methods, reference standards, and study design, together with the dual and context-dependent functions of individual miRNAs, limits comparability and hinders routine clinical adoption. Progress will depend on rigorous assay standardization, harmonized reporting, and validation of candidate miRNAs and panels in large, prospective, multi-centre cohorts. Integrating miRNA profiles with other molecular and clinical data, and advancing safe, effective miRNA-based therapeutics, will be essential to translate this promising biomarker class into tangible benefits for patients.

Abstract:

The selection of an optimal antifoam is critical for efficient fermentation, as industrial agents often have detrimental side effects like growth inhibition, while some can enhance productivity. This study presents a rational approach to developing and screening novel silicone-polyol antifoam emulsions. A key finding was the discovery of selective antibacterial activity in agent 3L10, which strongly inhibited Gram-positive bacteria (especially Corynebacterium glutamicum) but not Gram-negative strains. This specificity, likely mediated by interaction with the mycolic acid layer of C. glutamicum, highlights the necessity for strain-specific antifoam testing. A comprehensive evaluation protocol—combining chemical design, cytotoxicity screening across diverse microorganisms, determination of minimum effective concentrations (MEC), and validation in model bioreactor fermentations—was established. Through this process, agent 6T80 was identified as a promising candidate. It exhibited low MEC, high emulsion stability, no cytotoxicity, and did not impair growth or recombinant protein production in B. subtilis or P. putida fermentations. The study concludes that agent 6T80 is suitable for further application in processes involving Gram-negative and certain Gram-positive hosts, whereas agent 3L10 serves as a valuable tool for studying surfactant-membrane interactions. The developed methodology enables the targeted selection of highly efficient and biocompatible antifoams for specific biotechnological processes.

Abstract: 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a non-proteinogenic aromatic compound that functions as a key biosynthetic precursor for diverse secondary metabolites with pharmaceutical and industrial value. Microbial production of 3,4-AHBA offers a sustain-able alternative to petroleum-based chemical synthesis; however, metabolic complexity and trade-offs between growth and product formation constrain rational strain design. Here, genome-scale metabolic (GSM) modeling and flux balance analysis (FBA) were in-tegrated with targeted genetic engineering to elucidate and enhance 3,4-AHBA production in Streptomyces thermoviolaceus. A genome-scale metabolic model was constructed and ex-panded by incorporating the nspH–nspI gene operon, which encodes the 3,4-AHBA bio-synthetic pathway. In silico FBA predicted substantial rewiring of central carbon metabo-lism, with carbon flux redirected from glycolysis and the tricarboxylic acid cycle toward aspartate-derived intermediates and 3,4-AHBA synthesis, accompanied by reduced bio-mass-associated flux. Guided by these predictions, an engineered strain (St::NspHI) was developed and experimentally evaluated. Consistent with model predictions, the engi-neered strain exhibited lower growth rates and glucose uptake than the wild type, reflect-ing a metabolic burden. Nevertheless, 3,4-AHBA production was achieved exclusively in the engineered strain. Comparison of simulated and experimental fluxes revealed overes-timation by FBA, likely due to secondary metabolism and incomplete genome annotation. Overall, GSM-guided design enables optimization of precursor production.

Abstract: Oleaginous yeasts are promising microbial platforms for lipid production from non-conventional carbon sources; however, acetate utilization is frequently constrained by physiological limitations associated with culture pH. In this study, acetate utilization, biomass formation, and lipid production by Lipomyces starkeyi were investigated under flask and fed-batch cultivation to evaluate the influence of culture pH and pH control strategy. Statistically supported flask-scale experiments demonstrated that acetate concentration and cultivation time significantly affected acetate consumption, biomass formation, lipid yield, and culture pH, with excessive acetate loading resulting in culture alkalization, incomplete substrate utilization, and reduced process performance. Although lipid yield increased with increasing acetate concentration, lipid content and fatty acid composition remained unchanged, indicating that enhanced lipid production was primarily attributable to increased biomass formation rather than to changes in lipid biosynthesis. Fed-batch cultivation under different pH-control strategies provided qualitative insights into the relationships among pH regulation, acetate availability, and lipid accumulation under controlled fermentation conditions. While lipid accumulation was observed under both HCl-based and acetic acid–based pH control, differences in pH stability and cumulative acetate availability were associated with distinct patterns of lipid production. Collectively, these results identify culture pH as a critical physiological parameter influencing acetate utilization and lipid accumulation in L. starkeyi and suggest that coordinated pH control and carbon feeding strategies may improve the robustness of acetate-based lipid production processes. Further replicated fed-batch studies will be required to quantitatively validate these trends and support industrial applications.

Abstract: The rapid expansion of probiotics and other microbiome-modulating interventions has been accompanied by a growing number of human clinical trials. Yet, despite frequent reports of statistically significant microbiome changes, relatively few studies generate evidence that convincingly supports health claims or translates into reproducible, clinically meaningful outcomes. This gap is often attributed to the inherent complexity and inter-individual variability of the gut microbiome; however, recurring shortcomings in trial design and interpretation likely play an equally important role.In this Commentary, we examine common failure modes that weaken the clinical validation of microbiome-mediated interventions. These include overreliance on descriptive microbiome metrics (e.g., alpha diversity and taxonomic shifts) as surrogate endpoints, misalignment between prespecified endpoints and the claims ultimately advanced, and excessive dependence on symptom-only outcomes in settings characterized by substantial placebo responsiveness. We further highlight how inadequate control of key confounders—particularly diet, antibiotic exposure, and concomitant medications—combined with endpoint overload and underpowered study designs, can obscure true biological signal and increase the risk of irreproducible findings.We argue that stronger evidence emerges when the microbiome is treated as a mechanistic mediator rather than a clinical endpoint. Trials are most interpretable when intended claims are prospectively defined, linked to explicit biological mechanisms, and evaluated using a hierarchy of endpoints that prioritizes host-relevant outcomes and objective biomarkers, with microbiome measures integrated to support mechanistic plausibility. Adoption of staged development pathways, disciplined statistical planning, and transparent management of confounding variables can further improve reproducibility and clinical relevance.

Abstract: Background: Microbiome-targeted interventions have shown promise for metabolic health, yet clinical evidence remains inconsistent, particularly across stages of metabolic disease. This study evaluated the metabolic effects, safety, and tolerability of EDC-HHA01, a microbiome-informed, non-pharmacologic intervention, in adults with prediabetes (PD) or Type 2 Diabetes (T2DM). Methods: In a randomized, double-blind, placebo-controlled clinical trial, participants received EDC-HHA01 or placebo for six months. The study was adequately powered (≥80%) for the primary endpoint. Outcomes included changes in glycated hemoglobin (HbA1c), indices of insulin resistance, markers of metabolic endotoxemia, safety-related laboratory parameters, and exploratory patient-reported measures. Analyses were stratified by metabolic status and background metformin use. Results: In participants with PD, EDC-HHA01 supplementation was associated with a statistically and clinically meaningful reduction in HbA1c compared with placebo, supported by concordant improvements in fasting insulin, insulin resistance indices, and reductions in endotoxemia markers. In participants with T2DM, changes were directionally similar but attenuated and did not reach statistical significance. The intervention was well tolerated, with no serious adverse events, high adherence, and no clinically relevant adverse changes in renal or lipid parameters. Exploratory patient-reported outcomes indicated favorable acceptability but were not interpreted as efficacy endpoints. Conclusions: EDC-HHA01 was associated with biologically coherent, stage-dependent metabolic effects, most evident in PD. These findings support further investigation of microbiome-informed strategies as metabolic support in early-stage dysregulation.