Project description:In our rabbit model of pulmonary tuberculosis, infection with Mtb HN878, a hyper-virulent W-Beijing strain, results in progressive cavitary disease. However, infection of rabbit lungs with Mtb CDC1551, a hyper-immunogenic strain is effectively controlled overtime, establishing latent Mtb infection. Using these two Mtb strains, we tested the hypothesis that the initial host response in the lungs within hours of infection determines later outcome. The microarray experiments was performed to identify gene expression changes in the Mtb-HN878 or CDC1551- infected rabbit lungs at 3 hours post infection, compared to uninfected naïve rabbit lungs. New Zealand White rabbits were infected with Mtb HN878 or CDC1551 at ~3.5log10. At 3 hours post infection, lung tissue from Mtb-infected and uninfected rabbits were isolated and used for total RNA extraction. Total rabbit lung RNA was used for microarray analysis to determine infection induced changes in host gene expression.
Project description:We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of (1) Beijing/W and non-Beijing/W clinical strains, and (2) susceptible and multidrug-resistant (MDR-) MTB strains. THP-1 cells were induced differentiation into a macrophage phenotype. Then cells were infected with three MDR (INHR, RIFR) Beijing/W, three sensitive (INHS, RIFS) Beijing/W, three MDR(INHR, RIFR) non-Beijing/W, and three sensitive (INHS, RIFS) non-Beijing/W strains. Total RNA were extracted and transfered into cDNA for miRNA profile analysis. Non-infected cells were used as control.
Project description:Chromosomal copy number variations(CNV) have been associated with various neurological and developmental disorders and chromosomal microarray (CMA) is a method of choice to diagnose Copy Number Gain/Loss syndromes. Recently, next-generation sequencing (NGS)-based low-coverage whole genome sequencing (LC-WGS) has been applied to detect Copy Number Gain/Loss syndromes. This dataset is intended to be used as a “Golden standard data set” for development of LC-WGS analysis method. It consists of patients (n=63) who have a mental delay and/or physical disability phenotype and normal (n=20) phenotype. Overall design: CNV analysis using Affymetrix 750K and HD array was performed on 63 samples with developmental delay and/or physical disability phenotype. There are also 20 samples who were not diagnostic of any CNV disease and normal phenotype.
Project description:New tuberculosis vaccines are highly desirable and urgently needed since the attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) provides only variable efficacy against the pulmonary form of the disease. The region of difference 1 (RD1), which is deleted in BCG and strongly impacts on Mycobacterium tuberculosis (Mtb) virulence and immunogenicity, represents a crucial locus to be engineered for either the improvement of the current BCG vaccine, or the attenuation of Mtb. Therefore, mutants secreting or not wild-type or mutated variants of the RD1-encoded 6 kDa early secreted antigenic target (ESAT-6) were generated. Comparative analysis of the transcriptome, phenotype, cytokine production profiles and the capacity to promote T cell responses were conducted in human primary dendritic cells (DCs), as they represent critical regulators of vaccine-induced immunity, unveiling a distinct immunogenic potential for BCG or Mtb mutants. In contrast to Mtb, BCG induced a poor DC maturation, and to our surprise, a BCG strain complemented with the RD1 region only partially restored DC maturation and expansion of interferon (IFN)-γ producing T cells. In contrast, infection with a recombinant attenuated Mtb strain, secreting a truncated version of ESAT-6 lacking 11 amino acids at the C-terminus portion, drove full maturation in infected DC and maintained their capacity to promote polarization of T helper (Th) 1 cells, as observed upon infection with the virulent Mtb. We performed a comparative microarray analysis of dendritic cells (DCs), infected with Mtb and BCG strains, expressing/complemented (MtbΔRD1::RD1 and BCG::RD1) or not (MtbΔRD1::B412 and BCG::B412) the/with the RD1 region. DCs were challenged with different BCG and Mtb recombinant strains for 8h.
Project description:Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. In this work, we have identified the SigE regulon during infection of macrophages Our results indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function (i.e., detoxification and secretion). Keywords: strains comparison We compared the global gene expression of the H37Rv strain and the sigE mutant strain of Mtb in different conditions: growing exponentially in liquid media; after 2h of incubation in RPMI; or after 24 hours of infection of human macrophage-like THP-1 cells.
Project description:Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. In this work, we have identified the SigE regulon during infection of macrophages Our results indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function (i.e., detoxification and secretion). Keywords: strains comparison Overall design: We compared the global gene expression of the H37Rv strain and the sigE mutant strain of Mtb in different conditions: growing exponentially in liquid media; after 2h of incubation in RPMI; or after 24 hours of infection of human macrophage-like THP-1 cells.
Project description:The main project purpose is to investigate the fundamental physiological state of M. tuberculosis (MTB) during the infection and the mycobacterial response within the infected host tissue, human lung. High-throughput proteomic analysis of MTB cells will be involved to solve this problem. The description of pattern of proteins expressed in MTB cells extracted directly from clinical material of patients with tuberculosis defines the main novelty of the given project. Undoubtedly, the intermediate project results describing the features of MTB strains proteome in vitro will also be essential for the global scientific community.
Project description:Amox-Clav combination downregulates the expression of WhiB4. Further, MtbΔwhiB4 survives better upon treatment with this combination. Therefore, to study the role of WhiB4 in regulating the response of Mtb to this combination, we carried out global transcriptome profiling of Wt Mtb and MtbΔwhiB4 strains after treatment. Overall design: Mtb H37Rv and MtbΔwhiB4 were grown till early log phase and treated with 10X Augmentin at 100 µg/ml of Amoxicillin and 8 µg/ml of Clavulanate for 6 h at 37 degree at 150rpm. Wildtype Mtb H37Rv were treated with 1X Augmentin (10 µg/ml of Amoxicillin and 8 µg/ml of Clavulanate), or 5X Augmentin (50 µg/ml of Amoxicillin and 8 µg/ml of Clavulanate) for 6 or 12 h at 37 degree at 150rpm. Experiments were done in duplicate, triplicate, tetraplicate or pentaplicate.