Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

Two high-quality Prototheca zopfii genomes provide new insights into their evolution as obligate algal heterotrophs and their pathogenicity.

The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Prototheca zopfii Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although P. zopfii maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two P. zopfii genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of Prototheca and non-photosynthetic organisms. The two high-quality P. zopfii genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity. IMPORTANCE: The genus Prototheca, characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii. The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.

LRTK: a platform agnostic toolkit for linked-read analysis of both human genome and metagenome.

BACKGROUND: Linked-read sequencing technologies generate high-base quality short reads that contain extrapolative information on long-range DNA connectedness. These advantages of linked-read technologies are well known and have been demonstrated in many human genomic and metagenomic studies. However, existing linked-read analysis pipelines (e.g., Long Ranger) were primarily developed to process sequencing data from the human genome and are not suited for analyzing metagenomic sequencing data. Moreover, linked-read analysis pipelines are typically limited to 1 specific sequencing platform. FINDINGS: To address these limitations, we present the Linked-Read ToolKit (LRTK), a unified and versatile toolkit for platform agnostic processing of linked-read sequencing data from both human genome and metagenome. LRTK provides functions to perform linked-read simulation, barcode sequencing error correction, barcode-aware read alignment and metagenome assembly, reconstruction of long DNA fragments, taxonomic classification and quantification, and barcode-assisted genomic variant calling and phasing. LRTK has the ability to process multiple samples automatically and provides users with the option to generate reproducible reports during processing of raw sequencing data and at multiple checkpoints throughout downstream analysis. We applied LRTK on linked reads from simulation, mock community, and real datasets for both human genome and metagenome. We showcased LRTK's ability to generate comparative performance results from preceding benchmark studies and to report these results in publication-ready HTML document plots. CONCLUSIONS: LRTK provides comprehensive and flexible modules along with an easy-to-use Python-based workflow for processing linked-read sequencing datasets, thereby filling the current gap in the field caused by platform-centric genome-specific linked-read data analysis tools.

Effect of different ulnar osteotomies on loading of the distal radioulnar joint: a finite element analysis.

BACKGROUND: Ulnar impingement syndrome is a prevalent source of ulnar carpal pain; however, there is ongoing debate regarding the specific location of shortening, the method of osteotomy, the extent of shortening, and the resulting biomechanical alterations. METHOD: To investigate the biomechanical changes in the distal radioulnar joint (DRUJ) resulting from different osteotomy methods, a cadaveric specimen was dissected, and the presence of a stable DRUJ structure was confirmed. Subsequently, three-dimensional data of the specimen were obtained using a CT scan, and finite element analysis was conducted after additional processing. RESULTS: The DRUJ stress did not change significantly at the metaphyseal osteotomy of 2-3 mm but increased significantly when the osteotomy length reached 5 mm. When the osteotomy was performed at the diaphysis, the DRUJ stress increased with the osteotomy length, and the increase was greater than that of metaphyseal osteotomy. Stress on the DRUJ significantly increases when the position is changed to pronation dorsi-extension. Similarly, the increase in stress in diaphyseal osteotomy was greater than that in metaphyseal osteotomy. When the model was subjected to a longitudinal load of 100 N, neither osteotomy showed a significant change in DRUJ stress at the neutral position. However, the 100 N load significantly increased stress on the DRUJ when the position was changed to pronation dorsi-extension, and the diaphyseal osteotomy significantly increased stress on the DRUJ. CONCLUSIONS: For patients with distal oblique bundle, metaphyseal osteotomy result in a lower increase in intra-articular pressure in the DRUJ compared to diaphyseal osteotomy. However, it is crucial to note that regardless of the specific type of osteotomy employed, it is advisable to avoid a shortening length exceeding 5 mm.

Whole exome sequencing identified a homozygous novel variant in DOP1A gene in the Pakistan family with neurodevelopmental disabilities: case report and literature review.

Background: Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs. Case presentation: Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM_001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs). Conclusion: Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans.

Exploring high-quality microbial genomes by assembling short-reads with long-range connectivity.

Although long-read sequencing enables the generation of complete genomes for unculturable microbes, its high cost limits the widespread adoption of long-read sequencing in large-scale metagenomic studies. An alternative method is to assemble short-reads with long-range connectivity, which can be a cost-effective way to generate high-quality microbial genomes. Here, we develop Pangaea, a bioinformatic approach designed to enhance metagenome assembly using short-reads with long-range connectivity. Pangaea leverages connectivity derived from physical barcodes of linked-reads or virtual barcodes by aligning short-reads to long-reads. Pangaea utilizes a deep learning-based read binning algorithm to assemble co-barcoded reads exhibiting similar sequence contexts and abundances, thereby improving the assembly of high- and medium-abundance microbial genomes. Pangaea also leverages a multi-thresholding algorithm strategy to refine assembly for low-abundance microbes. We benchmark Pangaea on linked-reads and a combination of short- and long-reads from simulation data, mock communities and human gut metagenomes. Pangaea achieves significantly higher contig continuity as well as more near-complete metagenome-assembled genomes (NCMAGs) than the existing assemblers. Pangaea also generates three complete and circular NCMAGs on the human gut microbiomes.

VCF2PCACluster: a simple, fast and memory-efficient tool for principal component analysis of tens of millions of SNPs.

Principal component analysis (PCA) is an important and widely used unsupervised learning method that determines population structure based on genetic variation. Genome sequencing of thousands of individuals usually generate tens of millions of SNPs, making it challenging for PCA analysis and interpretation. Here we present VCF2PCACluster, a simple, fast and memory-efficient tool for Kinship estimation, PCA and clustering analysis, and visualization based on VCF formatted SNPs. We implemented five Kinship estimation methods and three clustering methods for its users to choose from. Moreover, unlike other PCA tools, VCF2PCACluster possesses a clustering function based on PCA result, which enabling users to automatically and clearly know about population structure. We demonstrated the same accuracy but a higher performance of this tool in performing PCA analysis on tens of millions of SNPs compared to another popular PLINK2 software, especially in peak memory usage that is independent of the number of SNPs in VCF2PCACluster.

The study on synthesis and vitro hypolipidemic activity of novel berberine derivatives nitric oxide donors.

Berberine was used as the lead compound in the present study to design and synthesize novel berberine derivatives by splicing bromine bridges of different berberine carbon chain lengths coupled nitric oxide donors, and their lipid lowering activities were assessed in a variety of ways. This experiment synthesized 17 new berberine nitric oxide donor derivatives. Compared with berberine hydrochloride, most of the compounds exhibited certain glycerate inhibitory activity, and compounds 6a, 6b, 6d, 12b and 12d showed higher inhibitory activity than berberine, with 6a, 6b and 6d having significant inhibitory activity. In addition, compound 6a linked to furazolidone nitric oxide donor showed better NO release in experiments; In further mechanistic studies, we screened and got two proteins, PCSK9 and ACLY, and docked two proteins with 17 compounds, and found that most of the compounds bound better with ATP citrate lyase (ACLY), among which there may be a strong interaction between compound 6a and ACLY, and the interaction force was better than the target drug Bempedoic Acid, which meaning that 6a may exert hypolipidemic effects by inhibiting ACLY; moreover, we also found that 6a may had the better performance in gastrointestinal absorption, blood-brain barrier permeability, Egan, Muegge class drug principle model calculation and bioavailability.

Effects of flora deficiency on the structure and function of the large intestine.

The significant anatomical changes in large intestine of germ-free (GF) mice provide excellent material for understanding microbe-host crosstalk. We observed significant differences of GF mice in anatomical and physiological involving in enlarged cecum, thinned mucosal layer and enriched water in cecal content. Furthermore, integration analysis of multi-omics data revealed the associations between the structure of large intestinal mesenchymal cells and the thinning of the mucosal layer. Increased Aqp8 expression in GF mice may contribute to enhanced water secretion or altered hydrodynamics in the cecum. In addition, the proportion of epithelial cells, nutrient absorption capacity, immune function and the metabolome of cecum contents of large intestine were also significantly altered. Together, this is the first systematic study of the transcriptome and metabolome of the cecum and colon of GF mice, and these findings contribute to our understanding of the intricate interactions between microbes and the large intestine.

CropGS-Hub: a comprehensive database of genotype and phenotype resources for genomic prediction in major crops.

The explosive amount of multi-omics data has brought a paradigm shift both in academic research and further application in life science. However, managing and reusing the growing resources of genomic and phenotype data points presents considerable challenges for the research community. There is an urgent need for an integrated database that combines genome-wide association studies (GWAS) with genomic selection (GS). Here, we present CropGS-Hub, a comprehensive database comprising genotype, phenotype, and GWAS signals, as well as a one-stop platform with built-in algorithms for genomic prediction and crossing design. This database encompasses a comprehensive collection of over 224 billion genotype data and 434 thousand phenotype data generated from >30 000 individuals in 14 representative populations belonging to 7 major crop species. Moreover, the platform implemented three complete functional genomic selection related modules including phenotype prediction, user model training and crossing design, as well as a fast SNP genotyper plugin-in called SNPGT specifically built for CropGS-Hub, aiming to assist crop scientists and breeders without necessitating coding skills. CropGS-Hub can be accessed at https://iagr.genomics.cn/CropGS/.

Comparative genomic analysis reveals niche adaption of Lactobacillus acidophilus.

AIMS: Lactobacillus acidophilus has been extensively applied in plentiful probiotic products. Although several studies have been performed to investigate the beneficial characteristics and genome function of L. acidophilus, comparative genomic analysis remains scarce. In this study, we collected 74 L. acidophilus genomes from our gut bacterial genome collection and the public database and conducted a comprehensive comparative genomic analysis. METHODS AND RESULTS: This study revealed the potential correlation of the genomic diversity and niche adaptation of L. acidophilus from different perspectives. The pan-genome of L. acidophilus was found to be open, with metabolism, information storage, and processing genes mainly distributed in the core genome. Phage- and peptidase-associated genes were found in the genome of the specificity of animal-derived strains, which were related to the adaptation of the animal gut. SNP analysis showed the differences of the utilization of vitamin B12 in cellular of L. acidophilus strains from animal gut and others. CONCLUSIONS: This work provides new insights for the genomic diversity analysis of L. acidophilus and uncovers the ecological adaptation of the specific strains.

Single-cell and spatiotemporal transcriptomic analyses reveal the effects of microorganisms on immunity and metabolism in the mouse liver.

The gut-liver axis is a complex bidirectional communication pathway between the intestine and the liver in which microorganisms and their metabolites flow from the intestine through the portal vein to the liver and influence liver function. In a sterile environment, the phenotype or function of the liver is altered, but few studies have investigated the specific cellular and molecular effects of microorganisms on the liver. To this end, we constructed single-cell and spatial transcriptomic (ST) profiles of germ-free (GF) and specific-pathogen-free (SPF) mouse livers. Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) revealed that the ratio of most immune cells was altered in the liver of GF mice; in particular, natural killer T (NKT) cells, IgA plasma cells (IgAs) and Kupffer cells (KCs) were significantly reduced in GF mice. Spatial enhanced resolution omics sequencing (Stereo-seq) confirmed that microorganisms mediated the accumulation of Kupffer cells in the periportal zone. Unexpectedly, IgA plasma cells were more numerous and concentrated in the periportal vein in liver sections from SPF mice but less numerous and scattered in GF mice. ST technology also enables the precise zonation of liver lobules into eight layers and three patterns based on the gene expression level in each layer, allowing us to further investigate the effects of microbes on gene zonation patterns and functions. Furthermore, untargeted metabolism experiments of the liver revealed that the propionic acid levels were significantly lower in GF mice, and this reduction may be related to the control of genes involved in bile acid and fatty acid metabolism. In conclusion, the combination of sc/snRNA-seq, Stereo-seq, and untargeted metabolomics revealed immune system defects as well as altered bile acid and lipid metabolic processes at the single-cell and spatial levels in the livers of GF mice. This study will be of great value for understanding host-microbiota interactions.

Benchmarking multi-platform sequencing technologies for human genome assembly.

Genome assembly is a computational technique that involves piecing together deoxyribonucleic acid (DNA) fragments generated by sequencing technologies to create a comprehensive and precise representation of the entire genome. Generating a high-quality human reference genome is a crucial prerequisite for comprehending human biology, and it is also vital for downstream genomic variation analysis. Many efforts have been made over the past few decades to create a complete and gapless reference genome for humans by using a diverse range of advanced sequencing technologies. Several available tools are aimed at enhancing the quality of haploid and diploid human genome assemblies, which include contig assembly, polishing of contig errors, scaffolding and variant phasing. Selecting the appropriate tools and technologies remains a daunting task despite several studies have investigated the pros and cons of different assembly strategies. The goal of this paper was to benchmark various strategies for human genome assembly by combining sequencing technologies and tools on two publicly available samples (NA12878 and NA24385) from Genome in a Bottle. We then compared their performances in terms of continuity, accuracy, completeness, variant calling and phasing. We observed that PacBio HiFi long-reads are the optimal choice for generating an assembly with low base errors. On the other hand, we were able to produce the most continuous contigs with Oxford Nanopore long-reads, but they may require further polishing to improve on quality. We recommend using short-reads rather than long-reads themselves to improve the base accuracy of contigs from Oxford Nanopore long-reads. Hi-C is the best choice for chromosome-level scaffolding because it can capture the longest-range DNA connectedness compared to 10× linked-reads and Bionano optical maps. However, a combination of multiple technologies can be used to further improve the quality and completeness of genome assembly. For diploid assembly, hifiasm is the best tool for human diploid genome assembly using PacBio HiFi and Hi-C data. Looking to the future, we expect that further advancements in human diploid assemblers will leverage the power of PacBio HiFi reads and other technologies with long-range DNA connectedness to enable the generation of high-quality, chromosome-level and haplotype-resolved human genome assemblies.

Marine Dehalogenator and Its Chaperones: Microbial Duties and Responses in 2,4,6-Trichlorophenol Dechlorination.

Marine environments contain diverse halogenated organic compounds (HOCs), both anthropogenic and natural, nourishing a group of versatile organohalide-respiring bacteria (OHRB). Here, we identified a novel OHRB (Peptococcaceae DCH) with conserved motifs but phylogenetically diverse reductive dehalogenase catalytic subunit (RdhAs) from marine enrichment culture. Further analyses clearly demonstrate the horizontal gene transfer of rdhAs among marine OHRB. Moreover, 2,4,6-trichlorophenol (TCP) was dechlorinated to 2,4-dichlorophenol and terminated at 4-chlorophenol in culture. Dendrosporobacter and Methanosarcina were the two dominant genera, and the constructed and verified metabolic pathways clearly demonstrated that the former provided various substrates for other microbes, while the latter drew nutrients, but might provide little benefit to microbial dehalogenation. Furthermore, Dendrosporobacter could readily adapt to TCP, and sporulation-related proteins of Dendrosporobacter were significantly upregulated in TCP-free controls, whereas other microbes (e.g., Methanosarcina and Aminivibrio) became more active, providing insights into how HOCs shape microbial communities. Additionally, sulfate could affect the dechlorination of Peptococcaceae DCH, but not debromination. Considering their electron accessibility and energy generation, the results clearly demonstrate that bromophenols are more suitable than chlorophenols for the enrichment of OHRB in marine environments. This study will greatly enhance our understanding of marine OHRB (rdhAs), auxiliary microbes, and microbial HOC adaptive mechanisms.

PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

Gene therapy for cross-correction of somatic organs and the CNS in mucopolysaccharidosis II in rodents and non-human primates.

Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.

Assembly and analytical validation of a metagenomic reference catalog of human gut microbiota based on co-barcoding sequencing.

Human gut microbiota is associated with human health and disease, and is known to have the second-largest genome in the human body. The microbiota genome is important for their functions and metabolites; however, accurate genomic access to the microbiota of the human gut is hindered due to the difficulty of cultivating and the shortcomings of sequencing technology. Therefore, we applied the stLFR library construction method to assemble the microbiota genomes and demonstrated that assembly property outperformed standard metagenome sequencing. Using the assembled genomes as references, SNP, INDEL, and HGT gene analyses were performed. The results demonstrated significant differences in the number of SNPs and INDELs among different individuals. The individual displayed a unique species variation spectrum, and the similarity of strains within individuals decreased over time. In addition, the coverage depth analysis of the stLFR method shows that a sequencing depth of 60X is sufficient for SNP calling. HGT analysis revealed that the genes involved in replication, recombination and repair, mobilome prophages, and transposons were the most transferred genes among different bacterial species in individuals. A preliminary framework for human gut microbiome studies was established using the stLFR library construction method.

Microbiota-mediated shaping of mouse spleen structure and immune function characterized by scRNA-seq and Stereo-seq.

Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan. As the largest secondary lymphoid organ, the spleen has a wide range of immunological functions. To explore the role of microbiota in regulating and shaping the spleen, we employ scRNA-seq and Stereo-seq technologies based on germ-free (GF) mice to detect differences in tissue size, anatomical structure, cell types, functions, and spatial molecular characteristics. We identify 18 cell types, 9 subtypes of T cells, and 7 subtypes of B cells. Gene differential expression analysis reveals that the absence of microorganisms results in alterations in erythropoiesis within the red pulp region and congenital immune deficiency in the white pulp region. Stereo-seq results demonstrate a clear hierarchy of immune cells in the spleen, including marginal zone (MZ) macrophages, MZ B cells, follicular B cells and T cells, distributed in a well-defined pattern from outside to inside. However, this hierarchical structure is disturbed in GF mice. Ccr7 and Cxcl13 chemokines are specifically expressed in the spatial locations of T cells and B cells, respectively. We speculate that the microbiota may mediate the structural composition or partitioning of spleen immune cells by modulating the expression levels of chemokines.

Ruthenium doping: An effective strategy for boosting nitrite electroreduction to ammonia over titanium dioxide nanoribbon array.

Electrochemical reduction of nitrite (NO2-) not only removes NO2- contaminant but also produces high-added value ammonia (NH3). This process, however, needs efficient and selective catalysts for NO2--to-NH3 conversion. In this study, Ruthenium doped titanium dioxide nanoribbon array supported on Ti plate (Ru-TiO2/TP) is proposed as an efficient electrocatalyst for the reduction of NO2- to NH3. When operated in 0.1 M NaOH containing NO2-, such Ru-TiO2/TP achieves an ultra-large NH3 yield of 1.56 mmol h-1 cm-2 and a super-high Faradaic efficiency of 98.9%, superior to its TiO2/TP counterpart (0.46 mmol h-1 cm-2, 74.1%). Furthermore, the reaction mechanism is studied by theoretical calculation.

HDAC2 exacerbates rheumatoid arthritis progression via the IL-17-CCL7 signaling pathway.

Histone deacetylases (HDACs) have been reported to regulate the immune response in rheumatoid arthritis (RA). The current study aimed to explore key HDACs and their molecular mechanism in RA. First, the expression of HDAC1, HDAC2, HDAC3 and HDAC8 in RA synovial tissue was determined by qRT-PCR. The effects of HDAC2 on the proliferation, migration, invasion, and apoptosis of fibroblast-like synoviocytes (FLS) in vitro were studied. Furthermore, collagen-induced arthritis (CIA) rat models were established to evaluate the severity of arthritis in joints, and the levels of inflammatory factors were examined by immunohistochemistry staining, ELISA, and qRT-PCR. Transcriptome sequencing was used to screen differentially expressed genes (DEGs) with HDAC2 silencing in the synovial tissue of CIA rat, and downstream signaling pathways were predicted by enrichment analysis. The results showed that HDAC2 was highly expressed in the synovial tissue of RA patients and CIA rats. Overexpressed HDAC2 promoted FLS proliferation, migration, and invasion and inhibited FLS apoptosis in vitro, resulting in secretion of inflammatory factors and RA exacerbation in vivo. There were 176 DEGs, including 57 downregulated and 119 upregulated genes, after silencing HDAC2 in CIA rats. DEGs were primarily enriched in Platinum drug resistance, IL-17 as well as the PI3K-Akt signaling pathways. CCL7, which was implicated in the IL-17 signaling pathway, was downregulated after HDAC2 silencing. Furthermore, CCL7 overexpression aggravated the development of RA, which was demonstrated to be effectively attenuated by HDAC2 suppression. In conclusion, this study demonstrated that HDAC2 exacerbated the progression of RA by regulating the IL-17-CCL7 signaling pathway, suggesting that HDAC2 may be a promising therapeutic target for RA treatment.