Chronic PM2.5 exposure disrupts intestinal barrier integrity via microbial dysbiosis-triggered TLR2/5-MyD88-NLRP3 inflammasome activation.

BACKGROUND: PM2.5, a known public health risk, is increasingly linked to intestinal disorders, however, the mechanisms of its impact are not fully understood. PURPOSE: This study aimed to explore the impact of chronic PM2.5 exposure on intestinal barrier integrity and to uncover the underlying molecular mechanisms. METHODS: C57BL/6 J mice were exposed to either concentrated ambient PM2.5 (CPM) or filtered air (FA) for six months to simulate urban pollution conditions. We evaluated intestinal barrier damage, microbial shifts, and metabolic changes through histopathology, metagenomics, and metabolomics. Analysis of the TLR signaling pathway was also conducted. RESULTS: The mean concentration of PM2.5 in the CPM exposure chamber was consistently measured at 70.9 ± 26.8 µg/m³ throughout the study period. Our findings show that chronic CPM exposure significantly compromises intestinal barrier integrity, as indicated by reduced expression of the key tight junction proteins Occludin and Tjp1/Zo-1. Metagenomic sequencing revealed significant shifts in the microbial landscape, identifying 35 differentially abundant species. Notably, there was an increase in pro-inflammatory nongastric Helicobacter species and a decrease in beneficial bacteria, such as Lactobacillus intestinalis, Lactobacillus sp. ASF360, and Eubacterium rectale. Metabolomic analysis further identified 26 significantly altered metabolites commonly associated with intestinal diseases. A strong correlation between altered bacterial species and metabolites was also observed. For example, 4 Helicobacter species all showed positive correlations with 13 metabolites, including Lactate, Bile acids, Pyruvate and Glutamate. Additionally, increased expression levels of TLR2, TLR5, Myd88, and NLRP3 proteins were noted, and their expression patterns showed a strong correlation, suggesting a possible involvement of the TLR2/5-MyD88-NLRP3 signaling pathway. CONCLUSIONS: Chronic CPM exposure induces intestinal barrier dysfunction, microbial dysbiosis, metabolic imbalance, and activation of the TLR2/5-MyD88-NLRP3 inflammasome. These findings highlight the urgent need for intervention strategies to mitigate the detrimental effects of air pollution on intestinal health and identify potential therapeutic targets.

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

Functional consequences of a rare missense BARD1 c.403G>A germline mutation identified in a triple-negative breast cancer patient.

We identified a rare missense germline mutation in BARD1 (c.403G>A or p.Asp135Asn) as pathogenic using integrated genomics and transcriptomics profiling of germline and tumor samples from an early-onset triple-negative breast cancer patient who later was administrated with a PARP inhibitor for 2 months. We demonstrated in cell and mouse models that, compared to the wild-type, (1) c.403G>A mutant cell lines were more sensitive to irradiation, a DNA damage agent, and a PARP inhibitor; (2) c.403G>A mutation inhibited interaction between BARD1 and RAD51 (but not BRCA1); and (3) c.403G>A mutant mice were hypersensitive to ionizing radiation. Our study shed lights on the clinical interpretation of rare germline mutations of BARD1.

RNA-seq analysis of testes from flurochloridone-treated rats.

Flurochloridone (FLC) is a widely used herbicide in developing countries. Although the testes are a target organ for FLC in rats, the adverse effects of FLC on testes have not been fully elucidated. To clarify them, we performed RNA-seq analysis using the testes of FLC-treated rats from our previous subchronic toxicity tests. Unilateral testes of three male rats from solvent control groupand three FLC-treated groups (3 mg/kg, 31.25 mg/kg and 125 mg/kg) were used for RNA extraction. A poly A selection protocol coupled with an Illumina TruSeq RNA-Seq library protocol was used to construct RNA-Seq libraries. Principal component analysis (PCA), differentially expressed gene (DEG) analysis, and hierarchical clustering analysis (HCA) were conducted using R. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to understand the biological characteristics of the DEGs using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The results indicated that many up-regulated DEGs were enriched in pathways associated with testicular injury, such as mitogen-activated protein kinase (MAPK) signaling, lysosome and focal adhesion. Many down-regulated DEGs were enriched in pathways associated with testicular reproduction function, such as sexual reproduction, spermatogenesis and germ cell development. Moreover, we confirmed the oral no-observed-adverse-effect level (NOAEL) of 3 mg/kg in subchronic toxicity test, because the overall testicular gene expression in 3 mg/kg FLC-treated group was similar to that of the solvent control group. In 31.25 mg/kg and 125 mg/kg groups, DEGs revealed that testicular injury was related to oxidative stress.

Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression.

Mitogen-activated protein kinase 1 (MAPK1) acts as an integration point for multiple biochemical signals, and is involved in a wide variety of biological processes such as cell proliferation and differentiation, transcription regulation, and development. Mitogen-activated protein kinase 1 plays an important role in inducing cell death in bacterial infections. In this study, the duck MAPK1 gene was cloned for the first time from the Cherry Valley duck. Sequence analysis showed that duck MAPK1 cDNA is 1,557 bp long, with an open reading frame of 1,107 bp. It encodes 368 amino acids, with 85.4, 84.5, and 97.3% homology with the human, mouse, and chicken MAPK1 gene, respectively. Furthermore, a SYBR Green quantitative real-time PCR assay was developed to detect duck MAPK1 expression. Following Riemerella anatipestifer infection by virulent strain Yb2, MAPK1 mRNA level increased more than 200-fold in the duck spleens, suggesting that increased duck MAPK1 expression can be used as an indicator of bacterial infection. Our results provide ground work to warrant further studies of the duck MAPK1 gene in bacterial pathogenesis.

Plasma-Free Blood as a Potential Alternative to Whole Blood for Transcriptomic Analysis.

RNA sequencing (RNAseq) technology has become increasingly important in precision medicine and clinical diagnostics, and emerged as a powerful tool for identifying protein-coding genes, performing differential gene analysis, and inferring immune cell composition. Human peripheral blood samples are widely used for RNAseq, providing valuable insights into individual biomolecular information. Blood samples can be classified as whole blood (WB), plasma, serum, and remaining sediment samples, including plasma-free blood (PFB) and serum-free blood (SFB) samples that are generally considered less useful byproducts during the processes of plasma and serum separation, respectively. However, the feasibility of using PFB and SFB samples for transcriptome analysis remains unclear. In this study, we aimed to assess the suitability of employing PFB or SFB samples as an alternative RNA source in transcriptomic analysis. We performed a comparative analysis of WB, PFB, and SFB samples for different applications. Our results revealed that PFB samples exhibit greater similarity to WB samples than SFB samples in terms of protein-coding gene expression patterns, detection of differentially expressed genes, and immunological characterizations, suggesting that PFB can serve as a viable alternative to WB for transcriptomic analysis. Our study contributes to the optimization of blood sample utilization and the advancement of precision medicine research. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00121-1.

Quartet metabolite reference materials for inter-laboratory proficiency test and data integration of metabolomics profiling.

BACKGROUND: Various laboratory-developed metabolomic methods lead to big challenges in inter-laboratory comparability and effective integration of diverse datasets. RESULTS: As part of the Quartet Project, we establish a publicly available suite of four metabolite reference materials derived from B lymphoblastoid cell lines from a family of parents and monozygotic twin daughters. We generate comprehensive LC-MS-based metabolomic data from the Quartet reference materials using targeted and untargeted strategies in different laboratories. The Quartet multi-sample-based signal-to-noise ratio enables objective assessment of the reliability of intra-batch and cross-batch metabolomics profiling in detecting intrinsic biological differences among the four groups of samples. Significant variations in the reliability of the metabolomics profiling are identified across laboratories. Importantly, ratio-based metabolomics profiling, by scaling the absolute values of a study sample relative to those of a common reference sample, enables cross-laboratory quantitative data integration. Thus, we construct the ratio-based high-confidence reference datasets between two reference samples, providing "ground truth" for inter-laboratory accuracy assessment, which enables objective evaluation of quantitative metabolomics profiling using various instruments and protocols. CONCLUSIONS: Our study provides the community with rich resources and best practices for inter-laboratory proficiency tests and data integration, ensuring reliability of large-scale and longitudinal metabolomic studies.

Integrated multiomic profiling of breast cancer in the Chinese population reveals patient stratification and therapeutic vulnerabilities.

Molecular profiling guides precision treatment of breast cancer; however, Asian patients are underrepresented in publicly available large-scale studies. We established a comprehensive multiomics cohort of 773 Chinese patients with breast cancer and systematically analyzed their genomic, transcriptomic, proteomic, metabolomic, radiomic and digital pathology characteristics. Here we show that compared to breast cancers in white individuals, Asian individuals had more targetable AKT1 mutations. Integrated analysis revealed a higher proportion of HER2-enriched subtype and correspondingly more frequent ERBB2 amplification and higher HER2 protein abundance in the Chinese HR+HER2+ cohort, stressing anti-HER2 therapy for these individuals. Furthermore, comprehensive metabolomic and proteomic analyses revealed ferroptosis as a potential therapeutic target for basal-like tumors. The integration of clinical, transcriptomic, metabolomic, radiomic and pathological features allowed for efficient stratification of patients into groups with varying recurrence risks. Our study provides a public resource and new insights into the biology and ancestry specificity of breast cancer in the Asian population, offering potential for further precision treatment approaches.

Extend the benchmarking indel set by manual review using the individual cell line sequencing data from the Sequencing Quality Control 2 (SEQC2) project.

Accurate indel calling plays an important role in precision medicine. A benchmarking indel set is essential for thoroughly evaluating the indel calling performance of bioinformatics pipelines. A reference sample with a set of known-positive variants was developed in the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, but the known indels in the known-positive set were limited. This project sought to provide an enriched set of known indels that would be more translationally relevant by focusing on additional cancer related regions. A thorough manual review process completed by 42 reviewers, two advisors, and a judging panel of three researchers significantly enriched the known indel set by an additional 516 indels. The extended benchmarking indel set has a large range of variant allele frequencies (VAFs), with 87% of them having a VAF below 20% in reference Sample A. The reference Sample A and the indel set can be used for comprehensive benchmarking of indel calling across a wider range of VAF values in the lower range. Indel length was also variable, but the majority were under 10 base pairs (bps). Most of the indels were within coding regions, with the remainder in the gene regulatory regions. Although high confidence can be derived from the robust study design and meticulous human review, this extensive indel set has not undergone orthogonal validation. The extended benchmarking indel set, along with the indels in the previously published known-positive set, was the truth set used to benchmark indel calling pipelines in a community challenge hosted on the precisionFDA platform. This benchmarking indel set and reference samples can be utilized for a comprehensive evaluation of indel calling pipelines. Additionally, the insights and solutions obtained during the manual review process can aid in improving the performance of these pipelines.

Correcting batch effects in large-scale multiomics studies using a reference-material-based ratio method.

BACKGROUND: Batch effects are notoriously common technical variations in multiomics data and may result in misleading outcomes if uncorrected or over-corrected. A plethora of batch-effect correction algorithms are proposed to facilitate data integration. However, their respective advantages and limitations are not adequately assessed in terms of omics types, the performance metrics, and the application scenarios. RESULTS: As part of the Quartet Project for quality control and data integration of multiomics profiling, we comprehensively assess the performance of seven batch effect correction algorithms based on different performance metrics of clinical relevance, i.e., the accuracy of identifying differentially expressed features, the robustness of predictive models, and the ability of accurately clustering cross-batch samples into their own donors. The ratio-based method, i.e., by scaling absolute feature values of study samples relative to those of concurrently profiled reference material(s), is found to be much more effective and broadly applicable than others, especially when batch effects are completely confounded with biological factors of study interests. We further provide practical guidelines for implementing the ratio based approach in increasingly large-scale multiomics studies. CONCLUSIONS: Multiomics measurements are prone to batch effects, which can be effectively corrected using ratio-based scaling of the multiomics data. Our study lays the foundation for eliminating batch effects at a ratio scale.

Quartet DNA reference materials and datasets for comprehensively evaluating germline variant calling performance.

BACKGROUND: Genomic DNA reference materials are widely recognized as essential for ensuring data quality in omics research. However, relying solely on reference datasets to evaluate the accuracy of variant calling results is incomplete, as they are limited to benchmark regions. Therefore, it is important to develop DNA reference materials that enable the assessment of variant detection performance across the entire genome. RESULTS: We established a DNA reference material suite from four immortalized cell lines derived from a family of parents and monozygotic twins. Comprehensive reference datasets of 4.2 million small variants and 15,000 structural variants were integrated and certified for evaluating the reliability of germline variant calls inside the benchmark regions. Importantly, the genetic built-in-truth of the Quartet family design enables estimation of the precision of variant calls outside the benchmark regions. Using the Quartet reference materials along with study samples, batch effects are objectively monitored and alleviated by training a machine learning model with the Quartet reference datasets to remove potential artifact calls. Moreover, the matched RNA and protein reference materials and datasets from the Quartet project enables cross-omics validation of variant calls from multiomics data. CONCLUSIONS: The Quartet DNA reference materials and reference datasets provide a unique resource for objectively assessing the quality of germline variant calls throughout the whole-genome regions and improving the reliability of large-scale genomic profiling.

Quartet RNA reference materials improve the quality of transcriptomic data through ratio-based profiling.

Certified RNA reference materials are indispensable for assessing the reliability of RNA sequencing to detect intrinsically small biological differences in clinical settings, such as molecular subtyping of diseases. As part of the Quartet Project for quality control and data integration of multi-omics profiling, we established four RNA reference materials derived from immortalized B-lymphoblastoid cell lines from four members of a monozygotic twin family. Additionally, we constructed ratio-based transcriptome-wide reference datasets between two samples, providing cross-platform and cross-laboratory 'ground truth'. Investigation of the intrinsically subtle biological differences among the Quartet samples enables sensitive assessment of cross-batch integration of transcriptomic measurements at the ratio level. The Quartet RNA reference materials, combined with the ratio-based reference datasets, can serve as unique resources for assessing and improving the quality of transcriptomic data in clinical and biological settings.

Multi-omics data integration using ratio-based quantitative profiling with Quartet reference materials.

Characterization and integration of the genome, epigenome, transcriptome, proteome and metabolome of different datasets is difficult owing to a lack of ground truth. Here we develop and characterize suites of publicly available multi-omics reference materials of matched DNA, RNA, protein and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters. These references provide built-in truth defined by relationships among the family members and the information flow from DNA to RNA to protein. We demonstrate how using a ratio-based profiling approach that scales the absolute feature values of a study sample relative to those of a concurrently measured common reference sample produces reproducible and comparable data suitable for integration across batches, labs, platforms and omics types. Our study identifies reference-free 'absolute' feature quantification as the root cause of irreproducibility in multi-omics measurement and data integration and establishes the advantages of ratio-based multi-omics profiling with common reference materials.

Correlation Analysis between GlpQ-Regulated Degradation of Wall Teichoic Acid and Biofilm Formation Triggered by Lactobionic Acid in Staphylococcus aureus.

Staphylococcus aureus biofilms are a serious problem in the food industry. Wall teichoic acid (WTA) is crucial in S. aureus biofilm formation. Overexpression of the WTA-hydrolyzing enzyme glycerophosphoryl diester phosphodiesterase (GlpQ), induced by lactobionic acid (LBA), may be related to biofilm formation. We investigated the relationship between the regulation on GlpQ degradation of WTA by LBA and S. aureus biofilm formation. LBA minimum inhibitory concentration for S. aureus was 12.5 mg/mL. Crystal violet staining revealed the LBA-mediated inhibition of S. aureus adhesion and biofilm formation. RT-qPCR revealed the repressed expression of adhesion-related genes by LBA. Scanning electron microscopy revealed the obvious disruption of S. aureus surface structure, confirming the repression of S. aureus adhesion and biofilm formation by LBA. Native-PAGE results suggested that the WTA content of S. aureus was reduced under the inhibition of LBA. Additionally, LBA induced the overexpression of glpQ. Combined with our previous work, these results suggest that glpQ is induced in S. aureus to function in WTA degradation with the addition of LBA, resulting in decreased WTA content and subsequent reduction of adhesion and biofilm formation. The findings provide new insight into the degradation mechanism of S. aureus WTA and indicate the potential of LBA as an anti-biofilm agent.

A comprehensive genomic and transcriptomic dataset of triple-negative breast cancers.

Molecular subtyping of triple-negative breast cancer (TNBC) is essential for understanding the mechanisms and discovering actionable targets of this highly heterogeneous type of breast cancer. We previously performed a large single-center and multiomics study consisting of genomics, transcriptomics, and clinical information from 465 patients with primary TNBC. To facilitate reusing this unique dataset, we provided a detailed description of the dataset with special attention to data quality in this study. The multiomics data were generally of high quality, but a few sequencing data had quality issues and should be noted in subsequent data reuse. Furthermore, we reconduct data analyses with updated pipelines and the updated version of the human reference genome from hg19 to hg38. The updated profiles were in good concordance with those previously published in terms of gene quantification, variant calling, and copy number alteration. Additionally, we developed a user-friendly web-based database for convenient access and interactive exploration of the dataset. Our work will facilitate reusing the dataset, maximize the values of data and further accelerate cancer research.

Comprehensive microRNA-seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats.

Rat is one of the most widely-used models in chemical safety evaluation and biomedical research. However, the knowledge about its microRNA (miRNA) expression patterns across multiple organs and various developmental stages is still limited. Here, we constructed a comprehensive rat miRNA expression BodyMap using a diverse collection of 320 RNA samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats with four biological replicates per group. Following the Illumina TruSeq Small RNA protocol, an average of 5.1 million 50 bp single-end reads was generated per sample, yielding a total of 1.6 billion reads. The quality of the resulting miRNA-seq data was deemed to be high from raw sequences, mapped sequences, and biological reproducibility. Importantly, aliquots of the same RNA samples have previously been used to construct the mRNA BodyMap. The currently presented miRNA-seq dataset along with the existing mRNA-seq dataset from the same RNA samples provides a unique resource for studying the expression characteristics of existing and novel miRNAs, and for integrative analysis of miRNA-mRNA interactions, thereby facilitating better utilization of rats for biomarker discovery.

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

BACKGROUND: Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. RESULTS: To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. CONCLUSIONS: Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.

Specific gene SEN1393 contributes to higher survivability of Salmonella Enteritidis in egg white by regulating sulfate assimilation pathway.

Salmonella enterica serovar Enteritidis (S. Enteritidis) presents an excellent capacity to survive in egg white, which is a hostile environment for bacterial growth. To reveal its survival mechanism, this study focuses on the specific gene SEN1393, which has been found to exist only in the genomic sequence of S. Enteritidis. The survival capacity of the deletion mutant strain ΔSEN1393 was proven to be significantly reduced after incubation in egg white. RNA sequencing and RT-qPCR results demonstrate that the expression levels of 19 genes were up-regulated, while the expression levels of 9 genes were down-regulated in egg white. These genes were classified into 6 groups based on their functional categories, namely the sulfate assimilation pathway, arginine biosynthesis, the tricarboxylic acid cycle, the fimbrial protein, the transport and chelation of metal ion, and others (sctT, rhs, and pspG). The strain ΔSEN1393 was deduced to damage FeS cluster enzymes and increase the sulfate and iron requirements, and to reduce bacterial motility and copper homeostasis. Via InterProScan analysis, the gene SEN1393 was speculated to encode a TerB-like and/or DjlA-like protein, and therefore, together with cysJ, possibly reduced the oxidative toxicities resulting from oxyanions such as tellurite, and/or improved CysPUWA conformation to restrain the uptake of the toxic oxyanions. In summary, the gene SEN1393 enabled the higher survival of S. Enteritidis in egg white as compared to other pathogens by regulating the sulfate assimilation pathway.

The homeoprotein Msx1 cooperates with Pkn1 to prevent terminal differentiation in myogenic precursor cells.

The homeoprotein Msx1 plays a critical role in embryonic patterning, particularly to maintain myogenic progenitor cell fate. However, the mechanisms underlying Msx1-mediated inhibition of myogenesis remain largely unknown. Here, we show that Msx1 cooperates with the protein kinase C-related kinase 1 (Pkn1), a member of the protein kinase C-related kinase family, to prevent the terminal differentiation of myogenic precursor cells. In mouse C2C12 cells, Pkn1 knockout partly impaired Msx1-mediated inhibition of myogenic differentiation, indicating a role for Pkn1 in this process. Furthermore, we found that Pkn1 was required for Msx1 enrichment at the promoter of Myf5, a myogenic regulatory gene. In Pkn1 knockout cells, this reduced Msx1 enrichment at the Myf5 promoter coincided with attenuated repression of Myf5 transcription. Together with our observation that Msx1 and Pkn1 were associated in a protein complex, these findings strongly suggest that Msx1 cooperates with Pkn1 to down-regulate Myf5 and, therefore, prevent the differentiation of myogenic precursor cells. Collectively, our data provide key insights into the mechanisms underlying Msx1 function in the prevention of myogenic differentiation.

Genomic and Transcriptomic Landscape of Triple-Negative Breast Cancers: Subtypes and Treatment Strategies.

We comprehensively analyzed clinical, genomic, and transcriptomic data of a cohort of 465 primary triple-negative breast cancer (TNBC). PIK3CA mutations and copy-number gains of chromosome 22q11 were more frequent in our Chinese cohort than in The Cancer Genome Atlas. We classified TNBCs into four transcriptome-based subtypes: (1) luminal androgen receptor (LAR), (2) immunomodulatory, (3) basal-like immune-suppressed, and (4) mesenchymal-like. Putative therapeutic targets or biomarkers were identified among each subtype. Importantly, the LAR subtype showed more ERBB2 somatic mutations, infrequent mutational signature 3 and frequent CDKN2A loss. The comprehensive profile of TNBCs provided here will serve as a reference to further advance the understanding and precision treatment of TNBC.