Post-acute sequelae of COVID-19 (PASC) disproportionately affect hospitalized patients and require improved molecular characterization to inform patient management. Here, we performed a prospective longitudinal multi-omics study of hospitalized COVID-19 patients, analyzing whole blood transcriptomics, targeted urine metabolomics, kidney injury biomarkers, and electronic health record-based outcome stratification across acute illness, one-month, and three-month recovery time points. Interconnected immunothrombosis-related pathways dominated the acute phase, while most immune and metabolomic pathways partially normalize. However, patients who developed long COVID exhibited a distinct blood transcriptional signature at three months consistent with an endothelial-associated activation profile, including platelet reactivity, complement dysregulation, and low-grade vascular inflammation, distinguishing them from fully recovered individuals. This multi-omics approach identifies clinically measurable biomarkers associated with longitudinal molecular trajectories and supports post-acute risk stratification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the Coronavirus Disease of 2019 (COVID-19) pandemic, can productively infect a variety of animal hosts. The evidence suggests that both wild and farmed animal populations supporting continuous transmission present us with novel concerning genotypes of SARS-CoV-2. This is especially worrisome within large and dense populations of farmed animals, such as minks, for which spillbacks of 'mink genotypes' to the human population have been recorded on numerous occasions. In this study, we present the results of continuous clinical, virological, serological, and genomic surveillance during the period of 11 months of the SARS-CoV-2 outbreak at the largest Latvian mink farm with ˃300 000 animals. Using the One Health approach, the COVID-19 status of minks and farm workers was constantly monitored during the surveillance period. The presence of the SARS-CoV-2 genome was confirmed in 299 minks and 32 farm workers during the outbreak. The phylogenetic analysis of 188 mink and 14 human SARS-CoV-2 isolates linked to the farm provided insight into the evolution of the virus in situ and contributed to epidemiological investigation. The results revealed that SARS-CoV-2 lineage B.1.177.60 was initially introduced to the mink farm by an infected farm worker between 17 February and 23 March 2021, and subsequently spread among the minks. Surveillance in the affected farm showed fluctuating virus circulation. Although the average seroprevalence in samples taken from live minks was 76.92%, a fluctuating course of infection was observed from April 2021 to March 2022. Despite the implementation of strict preventive and control measures at the farm, several additional SARS-CoV-2 strain introductions were identified during 11 months. The initial introduction of a common viral strain circulating among the people at the time soon resulted in the co-circulation of multiple sister sublineages that have evolved some concerning spike protein amino acid substitutions. Subsequent other lineage introductions into the farm were not able to spread among minks. Several independent cases of farm workers infected with genotypes restricted to this mink farm were documented throughout the study timeframe. However, the 'Latvian mink genotypes' were not detected in the general human population beyond epidemiologic association with the given mink farm.

BackgroundColorectal adenomas are key precancerous lesions and a major target for colorectal cancer prevention. While gut microbiome alterations are well described in colorectal cancer, microbial composition and functional capacity at the adenoma stage remain poorly understood. Emerging metagenomic data suggest early adenomas are associated with loss of microbial metabolic functions supporting epithelial and immune homeostasis.ObjectivesTo investigate the association between gut microbiome composition and functional pathways and the presence of colorectal adenomas in patients undergoing routine colonoscopy.Materials and methodsThis cross-sectional case-control study included adult patients undergoing routine colonoscopy. Participants were enrolled based on strict inclusion and exclusion criteria to minimize confounding factors such as inflammatory bowel disease, prior colorectal surgery, and recent antibiotic or probiotic use. Fecal samples were collected prior to bowel preparation, and gut microbiome taxonomic composition and functional pathways were analyzed using shotgun metagenomic sequencing.ResultsA total of 136 participants were included, of whom 56 had colorectal adenomas. Alpha diversity indices did not differ significantly between adenoma-positive and adenoma-negative groups. In contrast, beta diversity analysis revealed significant differences in overall microbial community structure. Descriptive genus-level differences suggested features of dysbiosis in adenoma-positive patients, including higher relative abundance of Bacteroides and Prevotella and lower abundance of Faecalibacterium and Anaerostipes. Differential abundance analysis identified a single species-level feature, UBA7597 sp003448195, enriched in the adenoma group. Functional profiling showed reduced microbial pathways related to menaquinone (vitamin K₂) biosynthesis, Stickland fermentation, and short-chain fatty acid (propionate) production in patients with adenomas.ConclusionsThe presence of colorectal adenomas was associated with reduced microbial metabolic functions linked to vitamin K₂ biosynthesis, amino acid fermentation, and propionate production, alongside compositional shifts toward a less functionally robust gut microbiome. These findings indicate that early colorectal neoplasia is accompanied by functional microbiome alterations that may serve as markers of adenoma-associated dysbiosis and provide insight into early metabolic changes in the colonic microenvironment.

Background Colorectal cancer (CRC) is among the most common malignancies worldwide, with colorectal adenomas recognized as well-established precursors to CRC. Changes in gut microbiota appear to be linked to CRC by promoting chronic inflammation, immune dysfunction, and metabolic issues that drive tumor growth and progression. Objectives To explore the relationship between gut microbiome composition and the presence of colorectal adenomas in patients undergoing routine colonoscopy. Materials and methods Patients were selected from those receiving standard colonoscopy based on strict inclusion and exclusion criteria, to minimize potential confounding factors such as previous colorectal surgeries, inflammatory bowel diseases, and the use of antibiotics or probiotics. Fecal samples were collected before bowel preparation for the colonoscopy procedure, and metagenomic shotgun sequencing was used to analyze the composition and functions of the gut microbiome. Results Overall, 136 participants were recruited, and 56 of them had colorectal adenomas. Although no distinction was observed in alpha diversity, beta diversity analysis indicated significant differences between the adenoma-positive and adenoma-negative groups. Signs of dysbiosis were found in patients with adenomas: increased abundance of the genera Bacteroides and Prevotella and decreased abundance of Faecalibacterium and Anaerostipes species. Beta diversity analysis showed statistically significant differences in the structure of the microbiota. Significant differences in the relative abundance of UBA7597 sp003448195 were observed between groups. Functionally, decreased vitamin K2, SCFA (propionate) synthesis, along with lower Stickland fermentation activity was observed, indicating altered microbial metabolism. These changes may compromise epithelial barrier support, anti-inflammatory signaling, and energy metabolism in the colon. Conclusions The discovery of microbial taxa and functional pathways associated with the presence of adenomas underscores the potential of microbiota-based biomarkers and therapeutic strategies in the prevention and management of colorectal cancer. ### Competing Interest Statement The authors have declared no competing interest. European Regional Development Fund, M-BM-^SSupport for applied researchM-BM-^T Project No.1.1.1.1/18/A/092 M-BM-^DRole of miRNAs in the host-gut microbiome communication during metformin treatment in the context of metabolic disordersM-BM-^T

BackgroundType 1 diabetes (T1D) is a multifactorial autoimmune disease mediated by genetic, epigenetic, and environmental factors. Diabetic kidney disease (DKD) is a major complication of diabetes mellitus which affects 30-40% of T1D patients. Increasing evidence suggests the significant role of the microbiome in the progression of both T1D and DKD.Materials and methodsHere we recruited 76 T1D patients and 22 healthy controls and combined data from sigmoid colon biopsy samples analysed with V3-V4 region amplification of 16S rRNA gene and shotgun metagenomics data obtained from faecal samples. Additionally, we compared T1D patients with and without progression of DKD.ResultsWe observed significant differences within both sample types at various taxonomic and functional levels. T1D patient microbiota detected using biopsy samples had a lower abundance of the Bacteroides genus when compared to healthy controls. Significantly, despite only a few taxonomic differences patients with and without DKD progression were vastly different at the functional pathway level within the faecal samples - we observed 2 and 61 enriched pathways in these groups. respectively, with several of these pathways linked to the mediation of renal function.ConclusionAltogether, we present novel data about microbial signatures relevant to T1D and DKD progression, which partly supports previous data and also presents possible tissue type or population-specific elements. DKD progression is characterized with significant differences within the functional level of the gut microbiome.

The gut microbiome composition is intricately linked to the host's health status, yet the mechanisms underlying its interaction with the host are not fully understood. MicroRNAs (miRNAs), facilitating intercellular communication, are found in bodily fluids, including the intestinal content, where they may affect the microbiome. However, their role in type 2 diabetes (T2D)-associated microbiome and treatment implications are not explored. Our study investigated how host miRNAs may influence gut microbiome changes related to metformin treatment in a T2D mouse model. Analyzing fecal and gut mucosal samples via small RNA sequencing, we correlated results with microbiome sequencing data, identifying miRNA-microbiome correlations, bacterial targets, and proteins targeted in these bacteria. Significant differences in miRNA expression based on diet and intestinal location were noted, with minor effects from metformin treatment in the proximal small intestine of non-diabetic male mice. Key fecal miRNAs targeting bacteria included mmu-miR-5119, mmu-miR-5126, mmu-miR-6538, and mmu-miR-2137, primarily affecting Oscillospiraceae_NOV, Lachnospiraceae_NOV, and Bacteroides. Our analysis of targeted proteins revealed diverse biological and molecular effects. Further research into miRNA-bacteria interactions could lead to new strategies for manipulating the gut microbiome in T2D and beyond.

Plant virus-like particles (VLPs) are highly ordered protein nanostructures with potential for biointerface engineering, targeted delivery, and templated nanomaterial synthesis. We investigated the self-assembly behavior and nucleic acid (NA) encapsulation capacity of coat proteins (CPs) from three sobemoviruses, focusing on ryegrass mottle virus (RGMoV) CP (RGCP). Expression in Pichia pastoris using a chromosome-integrated system enabled successful VLP formation, in contrast to the aggregate-prone assembly in Escherichia coli and Saccharomyces cerevisiae, likely due to the strong single-stranded DNS (ssDNA)-binding affinity of RGCP. RNAsequencing and RT-PCR confirmed selective encapsidation of CP mRNA, highlighting sequence-compactnessdriven self-packaging. Gel shift assays and a BacterioMatch II system revealed the preferential binding to ssDNA, which affected the assembly outcomes. Reassembly experiments with a CpG oligonucleotide demonstrated dose-dependent NA encapsulation and polymorphic capsid formation, with T = 1 and T = 3 symmetry particles forming at 1.5 mu g/mu l and T = 3 particles at 3 mu g/mu l. The observed structural transitions reflected the influence of NA size, secondary structure, pH, and potential CP truncation on capsid morphology. These findings suggested that RGMoV-derived VLPs are versatile and thermally stable bio-nanocontainers with tunable interior loading, offering novel strategies for constructing functional biointerfaces and nucleoprotein-based nanomaterials.

Arbuscular mycorrhizal fungi (AMF) colonize multiple plant hosts and form common mycelial networks (CMNs) that link multiple plants in nature. CMNs have been hypothesised to function as a highway for interplant information exchange to modulate plant biotic and abiotic stress responses. Here we used AMF Rhizophagus irregularis to inter connect two Medicago truncatula plants and explored the effect of known plant defence elicitor on pathogen tolerance of AMF connected inter-plant signal receivers. We analysed Medicago leaf metabolites (HPLC MS) and emitted volatiles (HS GS MS) together with RNA-seq data to compare responses of the inter-plant signal receivers with intact and cut CMN. The integrity of CMN significantly affected interplant signal receiver responses. Plant defence and signalling pathways were enriched with receiver transcripts that are uniquely changing in the intact vs interrupted CMN along with distinct production of plant isoprenoids: volatile monoterpenes and triterpene saponins. Furthermore, receivers of CMN-mediated signals from stressed senders display increased resistance to Fusarium sporotrichoides and susceptibility to Botrytis cinerea. Our results highlight CMN contribution to both upregulated and downregulated receiver plant gene functions and metabolites which may encode susceptibility and resistance factors that are important in pathogen species specific plant responses. Future dissection of the mechanisms involved in inter-plant signal decoding will yield novel discoveries on genetic regulation of interplant defence priming under pathogen attack. ### Competing Interest Statement The authors have declared no competing interest.

The gut microbiome plays a pivotal role in the modulation of host responses during viral infections, and recent studies have underscored its significance in the context of coronavirus disease 2019 (COVID-19). We aimed to investigate the dynamics and compositional changes in the gut microbiome of COVID-19 patients, addressing both the acute phase and the recovery process, with a particular focus on the emergence of post-COVID-19 conditions. Involving 146 COVID-19 patients and 110 healthy controls, this study employed a shotgun metagenomics approach for cross-sectional and longitudinal analyses with one- and three-month follow-ups. We observed a decline in taxonomic diversity among hospitalized COVID-19 patients compared to healthy controls, while a subsequent increase in alpha diversity was shown during the recovery process. A notable contribution of Enterococcus faecium was identified in the acute phase of the infection, accompanied by an increasing abundance of butyrate-producing bacteria (e.g., Roseburia, Lachnospiraceae_unclassified) during the recovery period. We highlighted a protective role of the Prevotella genus in the long-term recovery process and suggested a potential significance of population-specificity in the early gut microbiome markers of post-acute COVID-19 syndrome. Our study represents distinctive gut microbiome signatures in COVID-19, with potential diagnostic and prognostic implications, pinpointing potential modulators of the disease progression.

Metformin is widely used for treating type 2 diabetes mellitus (T2D). However, the efficacy of metformin monotherapy is highly variable within the human population. Understanding the potential indirect or synergistic effects of metformin on gut microbiota composition and encoded functions could potentially offer new insights into predicting treatment efficacy and designing more personalized treatments in the future. We combined targeted metabolomics and metagenomic profiling of gut microbiomes in newly diagnosed T2D patients before and after metformin therapy to identify potential pre-treatment biomarkers and functional signatures for metformin efficacy and induced changes in metformin therapy responders. Our sequencing data were largely corroborated by our metabolic profiling and identified that pre-treatment enrichment of gut microbial functions encoding purine degradation and glutamate biosynthesis was associated with good therapy response. Furthermore, we identified changes in glutamine-associated amino acid (arginine, ornithine, putrescine) metabolism that characterize differences in metformin efficacy before and after the therapy. Moreover, metformin Responders’ microbiota displayed a shifted balance between bacterial lipidA synthesis and degradation as well as alterations in glutamate-dependent metabolism of N-acetyl-galactosamine and its derivatives (e.g. CMP-pseudaminate) which suggest potential modulation of bacterial cell walls and human gut barrier, thus mediating changes in microbiome composition. Together, our data suggest that glutamine and associated amino acid metabolism as well as purine degradation products may potentially condition metformin activity via its multiple effects on microbiome functional composition and therefore serve as important biomarkers for predicting metformin efficacy.

Numerous type 2 diabetes (T2D) polygenic risk scores (PGSs) have been developed to predict individuals’ predisposition to the disease. An independent assessment and verification of the best-performing PGS are warranted to allow for a rapid application of developed models. To date, only 3% of T2D PGSs have been evaluated. In this study, we assessed all (n = 102) presently published T2D PGSs in an independent cohort of 3718 individuals, which has not been included in the construction or fine-tuning of any T2D PGS so far. We further chose the best-performing PGS, assessed its performance across major population principal component analysis (PCA) clusters, and compared it with newly developed population-specific T2D PGS. Our findings revealed that 88% of the published PGSs were significantly associated with T2D; however, their performance was lower than what had been previously reported. We found a positive association of PGS improvement over the years (p-value = 8.01 × 10−4 with PGS002771 currently showing the best discriminatory power (area under the receiver operating characteristic (AUROC) = 0.669) and PGS003443 exhibiting the strongest association PGS003443 (odds ratio (OR) = 1.899). Further investigation revealed no difference in PGS performance across major population PCA clusters and when compared with newly developed population-specific PGS. Our findings revealed a positive trend in T2D PGS performance, consistently identifying high-T2D-risk individuals in an independent European population.

The gut microbiome is a versatile system regulating numerous aspects of host metabolism. Among other traits, variations in the composition of gut microbial communities are related to blood lipid patterns and hyperlipidaemia, yet inconsistent association patterns exist. This study aims to assess the relationships between the composition of the gut microbiome and variations in lipid profiles among healthy adults. This study used data and samples from 23 adult participants of a previously conducted dietary intervention study. Circulating lipid measurements and whole-metagenome sequences of the gut microbiome were derived from 180 blood and faecal samples collected from eight visits distributed across an 11-week study. Lipid-related variables explained approximately 4.5% of the variation in gut microbiome compositions, with higher effects observed for total cholesterol and high-density lipoproteins. Species from the genera Odoribacter, Anaerostipes, and Parabacteroides correlated with increased serum lipid levels, whereas probiotic species like Akkermansia muciniphila were more abundant among participants with healthier blood lipid profiles. An inverse correlation with serum cholesterol was also observed for Massilistercora timonensis, a player in regulating lipid turnover. The observed correlation patterns add to the growing evidence supporting the role of the gut microbiome as an essential regulator of host lipid metabolism.

Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes.

Despite rapid improvements in the accessibility of whole-genome sequencing (WGS), understanding the extent of human genetic variation is limited by the scarce availability of genome sequences from underrepresented populations. Developing the population-scale reference database of Latvian genetic variation may fill the gap in European genomes and improve human genomics research. In this study, we analysed a high-coverage WGS dataset comprising 502 individuals selected from the Genome Database of the Latvian Population. An assessment of variant type, location in the genome, function, medical relevance, and novelty was performed, and a population-specific imputation reference panel (IRP) was developed. We identified more than 18.2 million variants in total, of which 3.3% so far are not represented in gnomAD and dbSNP databases. Moreover, we observed a notable though distinct clustering of the Latvian cohort within the European subpopulations. Finally, our findings demonstrate the improved performance of imputation of variants using the Latvian population-specific reference panel in the Latvian population compared to established IRPs. In summary, our study provides the first WGS data for a regional reference genome that will serve as a resource for the development of precision medicine and complement the global genome dataset, improving the understanding of human genetic variation.

Introduction. Although the presence of micro-organisms in the blood of healthy humans is a relatively new concept, there is a growing amount of evidence that blood might have its own microbiome.Gap Statement. Previous research has targeted the taxonomic composition of the blood microbiome using DNA-based sequencing methods, while little information is known about the presence of microbial transcripts obtained from the blood and their relation to conditions connected with increased gut permeability.Aim. To detect potentially alive and active micro-organisms and investigate differences in taxonomic composition between healthy people and patients with irritable bowel syndrome (IBS), we used the metatranscriptomics approach.Methodology. We collected blood samples from 23 IBS patients and 26 volunteers from the general population, and performed RNAseq on the isolated RNA. Reads corresponding to microbial genomes were identified with Kraken 2's standard plus protozoa and fungi database, and re-estimated at genus level with Bracken 2.7. We looked for trends in the taxonomic composition, making a comparison between the IBS and control groups, accounting for other different factors.Results. The dominant genera in the blood microbiome were found to be Cutibacterium, Bradyrhizobium, Escherichia, Pseudomonas, Micrococcus, Delftia, Mediterraneibacter, Staphylococcus, Stutzerimonas and Ralstonia. Some of these are typical environmental bacteria and could partially represent contamination. However, analysis of sequences from the negative controls suggested that some genera which are characteristic of the gut microbiome (Mediterraneibacter, Blautia, Collinsella, Klebsiella, Coprococcus, Dysosmobacter, Anaerostipes, Faecalibacterium, Dorea, Simiaoa, Bifidobacterium, Alistipes, Prevotella, Ruminococcus) are less likely to be a result of contamination. Differential analysis of microbes between groups showed that some taxa associated with the gut microbiome (Blautia, Faecalibacterium, Dorea, Bifidobacterium, Clostridium, Christensenella) are more prevalent in IBS patients compared to the general population. No significant correlations with any other factors were identified.Conclusion. Our findings support the existence of the blood microbiome and suggest the gut and possibly the oral microbiome as its origin, while the skin microbiome is a possible but less certain source. The blood microbiome is likely influenced by states of increased gut permeability such as IBS.

Introduction:Research findings of the past decade have highlighted the gut as the main site of action of the oral antihyperglycemic agent metformin despite its pharmacological role in the liver. Extensive evidence supports metformin's modulatory effect on the composition and function of gut microbiota, nevertheless, the underlying mechanisms of the host responses remain elusive. Our study aimed to evaluate metformin-induced alterations in the intestinal transcriptome profiles at different metabolic states.Methods:The high-fat diet-induced mouse model of obesity and insulin resistance of both sexes was developed in a randomized block experiment and bulk RNA-Seq of the ileum tissue was the method of choice for comparative transcriptional profiling after metformin intervention for ten weeks.Results:We found a prominent transcriptional effect of the diet itself with comparatively fewer genes responding to metformin intervention. The overrepresentation of immune-related genes was observed, including pronounced metformin-induced upregulation of immunoglobulin heavy-chain variable region coding Ighv1-7 gene in both high-fat diet and control diet-fed animals. Moreover, we provide evidence of the downregulation NF-kappa B signaling pathway in the small intestine of both obese and insulin-resistant animals as well as control animals after metformin treatment. Finally, our data pinpoint the gut microbiota as a crucial component in the metformin-mediated downregulation of NF-kappa B signaling evidenced by a positive correlation between the Rel and Rela gene expression levels and abundances of Parabacteroides distasonis, Bacteroides spp., and Lactobacillus spp. in the gut microbiota of the same animals.Discussion:Our study supports the immunomodulatory effect of metformin in the ileum of obese and insulin-resistant C57BL/6N mice contributed by intestinal immunoglobulin responses, with a prominent emphasis on the downregulation of NF-kappa B signaling pathway, associated with alterations in the composition of the gut microbiome.

Sobemovirus ryegrass mottle virus (RGMoV) is a single-stranded positive virus with a 30 nm viral particle size. It exhibits T=3 symmetry, with 180 coat protein (CP) subunits forming the virus structure. The RGMoV genome comprises five open reading frames, encoding P1, Px, a membrane-anchored 3C-like serine protease, a virus genome-linked protein, P16, an RNA-dependent RNA polymerase, and a coat protein. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was utilized to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, when the transcribed gRNA was introduced to oat plants, it failed to induce viral infection. This indicated the potential absence of certain sequences in either the 5’ or 3’ untranslated regions (UTR) or both. To resolve this, the complete sequence of the 3’ UTR was determined through 3’ end RACE, while the 5’ UTR was identified using high-throughput sequencing (HTS) - 5’ RACE-seq. Only the icDNA vector containing both newly identified UTR sequences proved infectious, resulting in classical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infection in oat plants after at least one passage. The successful generation of the icDNA highlights the synergistic potential of utilizing both methods when one approach alone fails. Furthermore, this study demonstrates the reliability of HTS as a method for determining the complete genome sequence of viral genomes.

The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-acquired enterobacteria is an important challenge for clinicians due to the limited therapeutic options for infections that are caused by these organisms. Here, we developed a panel of ESBL coding genes, evaluated the abundance and prevalence of ESBL encoding genes in patients undergoing H . pylori eradication therapy, and summarized the effects of eradication therapy on functional profiles of the gut microbiome. To assess the repertoire of known beta lactamase (BL) genes, they were divided into clusters according to their evolutionary relation. Primers were designed for amplification of cluster marker regions, and the efficiency of this amplification panel was assessed in 120 fecal samples acquired from 60 patients undergoing H . pylori eradication therapy. In addition, fecal samples from an additional 30 patients were used to validate the detection efficiency of the developed ESBL panel. The presence for majority of targeted clusters was confirmed by NGS of amplification products. Metagenomic sequencing revealed that the abundance of ESBL genes within the pool of microorganisms was very low. The global relative abundances of the ESBL-coding gene clusters did not differ significantly among treatment states. However, at the level of each cluster, classical ESBL producers such as Klebsiella sp. for bla OXY ( p = 0.0076), Acinetobacter sp. for bla ADC ( p = 0.02297) and others, differed significantly with a tendency to decrease compared to the pre- and post-eradication states. Only 13 clusters were common across all three datasets, suggesting a patient-specific distribution profile of ESBL-coding genes. The number of AMR genes detected in the post-eradication state was higher than that in the pre-eradication state, which could be attributed, at least in part, to the therapy. This study demonstrated that the ESBL screening panel was effective in targeting ESBL-coding gene clusters from bacterial DNA and that minor differences exist in the abundance and prevalence of ESBL-coding gene levels before and after eradication therapy.

Antidiabetic drug metformin alters the gut microbiome composition in the context of type 2 diabetes and other diseases; however, its effects have been mainly studied using fecal samples, which offer limited information about the intestinal site-specific effects of this drug. Our study aimed to characterize the spatial variation of the gut microbiome in response to metformin treatment by using a high-fat diet-induced type 2 diabetes mouse model of both sexes. Four intestinal parts, each at the luminal and mucosal layer level, were analyzed in this study by performing 16S rRNA sequencing covering six variable regions (V1-V6) of the gene and thus allowing to obtain in-depth information about the microbiome composition. We identified significant differences in gut microbiome diversity in each of the intestinal parts regarding the alpha and beta diversities. Metformin treatment altered the abundance of different genera in all studied intestinal sites, with the most pronounced effect in the small intestine, where Lactococcus increased remarkably. The abundance of Lactobacillus was substantially lower in male mice compared to female mice in all locations, in addition to an enrichment of opportunistic pathogens. Diet type and intestinal layer had significant effects on microbiome composition at each of the sites studied. We observed a different effect of metformin treatment on the analyzed subsets, indicating the multiple dimensions of metformin's effect on the gut microbiome.

Over the decades, practical biotechnology researchers have aimed to improve naturally occurring proteins and create novel ones. It is widely recognized that coupling protein sequence randomization with various effect screening methodologies is one of the most powerful techniques for quickly, efficiently, and purposefully acquiring these desired improvements. Over the years, considerable advancements have been made in this field. However, developing PCR-based or template-guided methodologies has been hampered by resultant template sequence biases. Here, we present a novel whole plasmid amplification-based approach, which we named OverFlap PCR, for randomizing virtually any region of plasmid DNA without introducing a template sequence bias.