Microbiome diversity in African American, European American, and Egyptian colorectal cancer patients

Purpose: Although there is an established role for microbiome dysbiosis in the pathobiology of colorectal cancer (CRC), CRC patients of various race/ethnicities demonstrate distinct clinical behaviors. Thus, we investigated microbiome dysbiosis in Egyptian, African American (AA), and European American (EA) CRC patients. Patients and methods: CRCs and their corresponding normal tissues from Egyptian (n = 17) patients of the Alexandria University Hospital, Egypt, and tissues from AA (n = 18) and EA (n = 19) patients at the University of Alabama at Birmingham were collected. DNA was isolated from frozen tissues, and the microbiome composition was analyzed by 16S rRNA sequencing. Differential microbial abundance, diversity, and metabolic pathways were identified using linear discriminant analysis (LDA) effect size analyses. Additionally, we compared these profiles with our previously published microbiome data derived from Kenyan CRC patients. Results:Differential microbiome analysis of CRCs across all racial/ethnic groups showed dysbiosis. There were high abundances of Herbaspirillum and Staphylococcus in CRCs of Egyptians, Leptotrichia in CRCs of AAs, Flexspiria and Streptococcus in CRCs of EAs, and Akkermansia muciniphila and Prevotella nigrescens in CRCs of Kenyans (LDA score \u3e4, adj. p-value Conclusions: Our findings showed altered mucosa-associated microbiome profiles of CRCs and their metabolic pathways across racial/ethnic groups. These findings provide a basis for future studies to link racial/ethnic microbiome differences with distinct clinical behaviors in CRC

Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells in the lamina propria of the small but not large intestine. By contrast, no changes in frequencies of CD4+CD25+FoxP3+ T cells were observed. Here we demonstrate that, surprisingly, the adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice significantly suppress antibodies directed against FIX. By contrast, equal numbers of splenic CD4+CD25+ T cells do not have an effect on antibody formation. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine and that suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg. The observation that CD4+CD25-LAP+ Treg migrate to the spleen are useful for the design of clinical protocols

Metagenome-Wide Associations and Metabolic Modeling to Predict the Biomarkers for Colorectal Cancer

Colorectal cancer (CRC) is the most common malignancy and the second most common cause of cancer-related deaths in the world. It is a multifactorial disease mediated by genetic, environmental, and lifestyle factors. Over time, the accumulation of genetic and epigenetic changes results in the activation of oncogenes and deactivation of tumor suppressor genes in the colon and rectal region which finally leads to tumor development. The gut microbiome is the main factor linking the effects of diet and environmental factors to host metabolism. Several studies showed the dysbiosis of gut microbiota in CRC patients with an increased proportion of pathogenic microbes and a reduction in the gut commensals. And it is also evident from the studies that pathogenic bacteria contribute to the CRC through DNA damage, pro-inflammatory signals, and altered signaling pathways. This strong link between the gut microbiome and CRC represents the gut microbiome as an ideal biomarker for CRC diagnosis and further harnesses it to develop new therapeutic interventions for CRC. Currently, the gut microbiome has gained immense importance in cancer research and human health. In this dissertation, a meta-analysis of shotgun metagenomic CRC datasets belonging to different geographical regions was performed using three distinct methods and identified 21 global microbial biomarkers for CRC. Co-occurrence network analysis of gut microbes showed higher co-occurrence correlations among the pathogenic species in CRC networks; in contrast, the control networks showed higher co-occurrences among gut commensals. Functional analysis of gut microbial communities showed variations in the enrichment of metabolic pathways and microbial contributors to the metabolic pathways between CRC and control gut communities. Analysis of butyrate synthesis pathways revealed the differences in the source of butanoate in control and CRC metagenomes. Pangenome gene family analysis of CRC-associated pathogenic species showed the high variability of these microbial species across the geographic regions, mainly attributed to differences in the accessory genes involved in bacterial virulence and antibiotic resistance. We further leveraged the predicted metabolome of microbial communities and showed the differences in metabolite composition of CRC and control groups. Our analysis also revealed altered metabolite and microbial interactions in CRC, reflecting the role of gut microbiota in CRC. Using metabolic modeling of CRC- and control-associated microbes on three different diet media identified the metabolic flux differences in isoprenoid and butyrate biosynthesis pathways in CRC and control communities. Overall, this study reported detailed taxonomic, functional, and metabolic differences between dysbiotic CRC and healthy control gut communities, which could serve as microbial and metabolite biomarkers for CRC. These findings could serve in understanding the dysbiosis in CRC and the development of non-invasive diagnostic and therapeutic strategies

StrainIQ: A Novel <em>n</em>-Gram-Based Method for Taxonomic Profiling of Human Microbiota at the Strain Level

The emergence of next-generation sequencing (NGS) technology has greatly influenced microbiome research and led to the development of novel bioinformatics tools to deeply analyze metagenomics datasets. Identifying strain-level variations in microbial communities is important to understanding the onset and progression of diseases, host–pathogen interrelationships, and drug resistance, in addition to designing new therapeutic regimens. In this study, we developed a novel tool called StrainIQ (strain identification and quantification) based on a new n-gram-based (series of n number of adjacent nucleotides in the DNA sequence) algorithm for predicting and quantifying strain-level taxa from whole-genome metagenomic sequencing data. We thoroughly evaluated our method using simulated and mock metagenomic datasets and compared its performance with existing methods. On average, it showed 85.8% sensitivity and 78.2% specificity on simulated datasets. It also showed higher specificity and sensitivity using n-gram models built from reduced reference genomes and on models with lower coverage sequencing data. It outperforms alternative approaches in genus- and strain-level prediction and strain abundance estimation. Overall, the results show that StrainIQ achieves high accuracy by implementing customized model-building and is an efficient tool for site-specific microbial community profiling

Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of &#216;C31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNAGln amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also

Transcriptome profile changes in the jejunum of nonhuman primates exposed to supralethal dose of total- or partial-body radiation

Abstract The risk of exposure of the general public or military personnel to high levels of ionizing radiation from nuclear weapons or radiological accidents is a dire national security matter. The development of advanced molecular biodosimetry methods, those that measure biological response, such as transcriptomics, to screen large populations of radiation-exposed victims is key to improving survival outcomes during radiological mass casualty scenarios. In this study, nonhuman primates were exposed to either 12.0 Gy cobalt-60 gamma (total-body irradiation, TBI) or X-ray (partial-body irradiation, PBI) 24 h after administration of a potential radiation medical countermeasure, gamma-tocotrienol (GT3). Changes in the jejunal transcriptomic profiles in GT3-treated and irradiated animals were compared to healthy controls to assess the extent of radiation damage. No major effect of GT3 on radiation-induced transcriptome at this radiation dose was identified. About 80% of the pathways with a known activation or repression state were commonly observed between both exposures. Several common pathways activated due to irradiation include FAK signaling, CREB signaling in the neurons, phagosome formation, and G-protein coupled signaling pathway. Sex-specific differences associated with excessive mortality among irradiated females were identified in this study, including Estrogen receptor signaling. Differential pathway activation was also identified across PBI and TBI, pointing towards altered molecular response for different degrees of bone marrow sparing and radiation doses. This study provides insight into radiation-induced changes in jejunal transcriptional profiles, supporting the investigation for the identification of biomarkers for radiation injury and countermeasure efficacy

Microbiome diversity in African American, European American, and Egyptian colorectal cancer patients

Purpose: Although there is an established role for microbiome dysbiosis in the pathobiology of colorectal cancer (CRC), CRC patients of various race/ethnicities demonstrate distinct clinical behaviors. Thus, we investigated microbiome dysbiosis in Egyptian, African American (AA), and European American (EA) CRC patients. Patients and methods: CRCs and their corresponding normal tissues from Egyptian (n = 17) patients of the Alexandria University Hospital, Egypt, and tissues from AA (n = 18) and EA (n = 19) patients at the University of Alabama at Birmingham were collected. DNA was isolated from frozen tissues, and the microbiome composition was analyzed by 16S rRNA sequencing. Differential microbial abundance, diversity, and metabolic pathways were identified using linear discriminant analysis (LDA) effect size analyses. Additionally, we compared these profiles with our previously published microbiome data derived from Kenyan CRC patients. Results: Differential microbiome analysis of CRCs across all racial/ethnic groups showed dysbiosis. There were high abundances of Herbaspirillum and Staphylococcus in CRCs of Egyptians, Leptotrichia in CRCs of AAs, Flexspiria and Streptococcus in CRCs of EAs, and Akkermansia muciniphila and Prevotella nigrescens in CRCs of Kenyans (LDA score >4, adj. p-value <0.05). Functional analyses showed distinct microbial metabolic pathways in CRCs compared to normal tissues within the racial/ethnic groups. Egyptian CRCs, compared to normal tissues, showed lower l-methionine biosynthesis and higher galactose degradation pathways. Conclusions: Our findings showed altered mucosa-associated microbiome profiles of CRCs and their metabolic pathways across racial/ethnic groups. These findings provide a basis for future studies to link racial/ethnic microbiome differences with distinct clinical behaviors in CRC