The thymus comprises an interconnected meshwork of epithelial cells (TECs) and thymocytes. This o... more The thymus comprises an interconnected meshwork of epithelial cells (TECs) and thymocytes. This organ is exceedingly sensitive to stress, undergoing rapid involution in response to infection, trauma, radiation exposure, and/or glucocorticoid treatments. The nature of the stress predicates whether the epithelial cells, developing thymocytes, and/or both are affected. MicroRNAs are small noncoding RNAs that regulate cellular responses to stress and during regeneration. We discovered that miR-205 is a stress responsive, epithelial-specific miR that modulates thymopoiesis. Mice lacking miR-205 selectively in TECs exhibited a significant decline in thymopoiesis beginning at 8 weeks of age. When stressed with a dsRNA mimic (polyI:C), these mice exhibited a much more severe thymic atrophy compared to littermate controls. Quantification of the TEC subsets revealed a defect in the proliferative recovery of the cortical TECs lacking miR-205. This contributed to a significant reduction in the ...
TCR-mediated intracellular signal transmission commences following the tyrosine phosphorylation o... more TCR-mediated intracellular signal transmission commences following the tyrosine phosphorylation of the CD3-encoded ITAMs. The CD3 ζ subunit, containing three ITAMs per chain, becomes one of the more heavily phosphorylated of the CD3 chains. The cytoplasmic tail of CD3 ζ contains multiple clusters of basic amino acids lysine and arginine. A number of transmembrane and cytosolic proteins, including the CD3 ϵ subunit, use these types of clusters to bind phospholipids. We report that the cytoplasmic tail of CD3 ζ contains a phospholipid-binding cluster with a relatively high affinity for PtdIns(3,5)P2, PtdIns(5)P, PtdIns(3)P, and PtdIns(4)P. The elimination of the phospholipid-binding function of CD3 ζ reduced the stability of TCR clustering at the immunological synapse. The function of this domain in TCR-mediated intracellular signaling responses will be described. These findings demonstrate a novel functional role for the CD3 ζ lipid-binding domain in T cell biology.
A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell... more A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell survival is described. Major histocompatibility complex class II–deficient mice were engineered to express Eα chains only in thymic epithelial cells in a tetracycline (tet)-controllable manner. This resulted in tet-responsive display of cell surface E complexes, positive selection of CD4+8– thymocytes, and generation of a CD4+ T cell compartment in a class II–barren periphery. Using this system, we have addressed two unresolved issues: the half-life of naive CD4+ T cells in the absence of class II molecules (3–4 wk) and the early signaling events associated with class II molecule engagement by naive CD4+ T cells (partial CD3 ζ chain phosphorylation and ZAP-70 association).
The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from... more The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3rd pharyngeal pouch during embryogenesis, this organ functions throughout life. Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less w...
Additional file 2: Fig. S1. DAP1 genetic association with SLE. Fig. S2. Genomic position of SLE a... more Additional file 2: Fig. S1. DAP1 genetic association with SLE. Fig. S2. Genomic position of SLE associated DAP1 variants. Fig. S3. DAP1 eQTL with rs2930047. Fig. S4. Autophagy induction data in PBMCs and monocytes. Fig. S5. Gene expression data. Fig. S6 Sm and snRNP antibodies in DAP1 SLE risk allele. Fig. S7. Heatmap of non-nuclear autoantigen signatures. Fig. S8 Expression of antigen presentation pathway. Fig. S9 DAP1 expression in primary B cells. Fig. S10 RNA-Seq data in PBMCs. Fig. S11. HAP3 defining key variants. Fig. S12. Statistical power analysis. Fig. S13. Uncropped blots.
Additional file 1: Table S1. DAP1-SLE association results based on Immunochipv.1. Table S2. List ... more Additional file 1: Table S1. DAP1-SLE association results based on Immunochipv.1. Table S2. List of sequencing variants in DAP1 gene region-European cohort. Table S3. 19 SLE associated DAP1 variants used for haplotype analysis. Table S4. List of variants in African-American and Chinese cohort. Table S5. Cochran-Mantel-Haenszel test result on SNPs. Table S6. DAP1 haplotype association analysis results. Table S7. Trans-ethnic analysis by Cochran-Mantel-Haenszel and MR-MEGA program. Table S8. Diplotypes analysis in Caucasian data. Table S9. Potentially regulatory variant annotations. Table S10- eQTL effects of SLE associated variants. Table S11. Lymphoblastoid Cell Lines (LCL) used in study. Table S12. PBMCs RNA-Seq analysis. Table S13. Reactome pathway analysis. Table S14. Autoantibody data. Table S15. List of participating institutions and investigators.
Purpose The human antibody repertoire forms in response to infections, the microbiome, vaccinatio... more Purpose The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. Methods An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. Results IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (...
The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tiss... more The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tissues in the body. Stress, in the form of infections, radiation exposure, and steroids, impairs thymic epithelial cell (TEC) functions and induces the programmed cell death of immature thymocytes. MicroRNAs are small noncoding RNAs involved in tissue repair and homeostasis, with several supporting T cell development. We report that miR-205, an epithelial-specific miR, maintains thymopoiesis following inflammatory perturbations. Thus, the activation of diverse pattern recognition receptors in mice causes a more severe thymic hypoplasia and delayed T cell recovery when miR-205 is conditionally ablated in TECs. Gene expression comparisons in the TECs with/without miR-205 revealed a significant differential regulation of chemokine/chemokine receptor pathways, antigen processing components, and changes in the Wnt signaling system. This was partly a consequence of reduced expression of the trans...
The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perfo... more The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-ζ chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of P...
The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a hetero... more The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR-zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.
Background Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease ch... more Background Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. Results We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells....
Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Whi... more Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 ... more Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50-350 nts in length and smaller RNAs (sncRNA) <50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists and locked nucleic acid sequence specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions rem...
Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and a... more Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and accurate assessment of the abundance and composition of the microbiome. While the impact of bead-beating on the recovery of OTUs at the phylum and class level have been studied, its influence on species-level microbiome recovery is not clear. Recent advances in sequencing technology has allowed species-level resolution of the microbiome using full length 16S rRNA gene sequencing instead of smaller amplicons that only capture a few hypervariable regions of the gene. We sequenced the v3-v4 hypervariable region as well as the full length 16S rRNA gene in mouse and human stool samples and discovered major clusters of gut bacteria that exhibit different levels of sensitivity to bead-beating treatment. Full length 16S rRNA gene sequencing unraveled vast species diversity in the mouse and human gut microbiome and enabled characterization of several unclassified OTUs in amplicon data. Many specie...
The thymus comprises an interconnected meshwork of epithelial cells (TECs) and thymocytes. This o... more The thymus comprises an interconnected meshwork of epithelial cells (TECs) and thymocytes. This organ is exceedingly sensitive to stress, undergoing rapid involution in response to infection, trauma, radiation exposure, and/or glucocorticoid treatments. The nature of the stress predicates whether the epithelial cells, developing thymocytes, and/or both are affected. MicroRNAs are small noncoding RNAs that regulate cellular responses to stress and during regeneration. We discovered that miR-205 is a stress responsive, epithelial-specific miR that modulates thymopoiesis. Mice lacking miR-205 selectively in TECs exhibited a significant decline in thymopoiesis beginning at 8 weeks of age. When stressed with a dsRNA mimic (polyI:C), these mice exhibited a much more severe thymic atrophy compared to littermate controls. Quantification of the TEC subsets revealed a defect in the proliferative recovery of the cortical TECs lacking miR-205. This contributed to a significant reduction in the ...
TCR-mediated intracellular signal transmission commences following the tyrosine phosphorylation o... more TCR-mediated intracellular signal transmission commences following the tyrosine phosphorylation of the CD3-encoded ITAMs. The CD3 ζ subunit, containing three ITAMs per chain, becomes one of the more heavily phosphorylated of the CD3 chains. The cytoplasmic tail of CD3 ζ contains multiple clusters of basic amino acids lysine and arginine. A number of transmembrane and cytosolic proteins, including the CD3 ϵ subunit, use these types of clusters to bind phospholipids. We report that the cytoplasmic tail of CD3 ζ contains a phospholipid-binding cluster with a relatively high affinity for PtdIns(3,5)P2, PtdIns(5)P, PtdIns(3)P, and PtdIns(4)P. The elimination of the phospholipid-binding function of CD3 ζ reduced the stability of TCR clustering at the immunological synapse. The function of this domain in TCR-mediated intracellular signaling responses will be described. These findings demonstrate a novel functional role for the CD3 ζ lipid-binding domain in T cell biology.
A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell... more A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell survival is described. Major histocompatibility complex class II–deficient mice were engineered to express Eα chains only in thymic epithelial cells in a tetracycline (tet)-controllable manner. This resulted in tet-responsive display of cell surface E complexes, positive selection of CD4+8– thymocytes, and generation of a CD4+ T cell compartment in a class II–barren periphery. Using this system, we have addressed two unresolved issues: the half-life of naive CD4+ T cells in the absence of class II molecules (3–4 wk) and the early signaling events associated with class II molecule engagement by naive CD4+ T cells (partial CD3 ζ chain phosphorylation and ZAP-70 association).
The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from... more The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3rd pharyngeal pouch during embryogenesis, this organ functions throughout life. Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less w...
Additional file 2: Fig. S1. DAP1 genetic association with SLE. Fig. S2. Genomic position of SLE a... more Additional file 2: Fig. S1. DAP1 genetic association with SLE. Fig. S2. Genomic position of SLE associated DAP1 variants. Fig. S3. DAP1 eQTL with rs2930047. Fig. S4. Autophagy induction data in PBMCs and monocytes. Fig. S5. Gene expression data. Fig. S6 Sm and snRNP antibodies in DAP1 SLE risk allele. Fig. S7. Heatmap of non-nuclear autoantigen signatures. Fig. S8 Expression of antigen presentation pathway. Fig. S9 DAP1 expression in primary B cells. Fig. S10 RNA-Seq data in PBMCs. Fig. S11. HAP3 defining key variants. Fig. S12. Statistical power analysis. Fig. S13. Uncropped blots.
Additional file 1: Table S1. DAP1-SLE association results based on Immunochipv.1. Table S2. List ... more Additional file 1: Table S1. DAP1-SLE association results based on Immunochipv.1. Table S2. List of sequencing variants in DAP1 gene region-European cohort. Table S3. 19 SLE associated DAP1 variants used for haplotype analysis. Table S4. List of variants in African-American and Chinese cohort. Table S5. Cochran-Mantel-Haenszel test result on SNPs. Table S6. DAP1 haplotype association analysis results. Table S7. Trans-ethnic analysis by Cochran-Mantel-Haenszel and MR-MEGA program. Table S8. Diplotypes analysis in Caucasian data. Table S9. Potentially regulatory variant annotations. Table S10- eQTL effects of SLE associated variants. Table S11. Lymphoblastoid Cell Lines (LCL) used in study. Table S12. PBMCs RNA-Seq analysis. Table S13. Reactome pathway analysis. Table S14. Autoantibody data. Table S15. List of participating institutions and investigators.
Purpose The human antibody repertoire forms in response to infections, the microbiome, vaccinatio... more Purpose The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. Methods An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. Results IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (...
The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tiss... more The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tissues in the body. Stress, in the form of infections, radiation exposure, and steroids, impairs thymic epithelial cell (TEC) functions and induces the programmed cell death of immature thymocytes. MicroRNAs are small noncoding RNAs involved in tissue repair and homeostasis, with several supporting T cell development. We report that miR-205, an epithelial-specific miR, maintains thymopoiesis following inflammatory perturbations. Thus, the activation of diverse pattern recognition receptors in mice causes a more severe thymic hypoplasia and delayed T cell recovery when miR-205 is conditionally ablated in TECs. Gene expression comparisons in the TECs with/without miR-205 revealed a significant differential regulation of chemokine/chemokine receptor pathways, antigen processing components, and changes in the Wnt signaling system. This was partly a consequence of reduced expression of the trans...
The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perfo... more The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-ζ chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of P...
The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a hetero... more The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR-zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.
Background Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease ch... more Background Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. Results We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells....
Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Whi... more Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 ... more Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50-350 nts in length and smaller RNAs (sncRNA) <50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists and locked nucleic acid sequence specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions rem...
Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and a... more Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and accurate assessment of the abundance and composition of the microbiome. While the impact of bead-beating on the recovery of OTUs at the phylum and class level have been studied, its influence on species-level microbiome recovery is not clear. Recent advances in sequencing technology has allowed species-level resolution of the microbiome using full length 16S rRNA gene sequencing instead of smaller amplicons that only capture a few hypervariable regions of the gene. We sequenced the v3-v4 hypervariable region as well as the full length 16S rRNA gene in mouse and human stool samples and discovered major clusters of gut bacteria that exhibit different levels of sensitivity to bead-beating treatment. Full length 16S rRNA gene sequencing unraveled vast species diversity in the mouse and human gut microbiome and enabled characterization of several unclassified OTUs in amplicon data. Many specie...
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