Molecular Diagnosis of Drug-Resistant Tuberculosis; A Literature Review.

Drug-resistant tuberculosis is a global health problem that hinders the progress of tuberculosis eradication programs. Accurate and early detection of drug-resistant tuberculosis is essential for effective patient care, for preventing tuberculosis spread, and for limiting the development of drug-resistant strains. Culture-based drug susceptibility tests are the gold standard method for the detection of drug-resistant tuberculosis, but they are time-consuming and technically challenging, especially in low- and middle-income countries. Nowadays, different nucleic acid-based assays that detect gene mutations associated with resistance to drugs used to treat tuberculosis are available. These tests vary in type and number of targets and in sensitivity and specificity. In this review, we will describe the available molecular tests for drug-resistant tuberculosis detection and discuss their advantages and limitations.

High-Resolution Chromatin Immunoprecipitation: ChIP-Sequencing.

Chromatin immunoprecipitation (ChIP) coupled with next-generation sequencing (NGS) is widely used for studying the nucleoprotein components that are involved in the various cellular processes required for shaping the bacterial nucleoid. This methodology, termed ChIP-sequencing (ChIP-seq), enables the identification of the DNA targets of DNA binding proteins across genome-wide maps. Here, we describe the steps necessary to obtain short, specific, high-quality immunoprecipitated DNA prior to DNA library construction for NGS and high-resolution ChIP-seq data.

Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica.

We previously developed a fermentation protocol for lipid accumulation in the oleaginous yeast Y. lipolytica. This process was used to perform transcriptomic time-course analyses to explore gene expression in Y. lipolytica during the transition from biomass production to lipid accumulation. In this experiment, a biomass concentration of 54.6 g(CDW)/l, with 0.18 g/g(CDW) lipid was obtained in ca. 32 h, with low citric acid production. A transcriptomic profiling was performed on 11 samples throughout the fermentation. Through statistical analyses, 569 genes were highlighted as differentially expressed at one point during the time course of the experiment. These genes were classified into 9 clusters, according to their expression profiles. The combination of macroscopic and transcriptomic profiles highlighted 4 major steps in the culture: (i) a growth phase, (ii) a transition phase, (iii) an early lipid accumulation phase, characterized by an increase in nitrogen metabolism, together with strong repression of protein production and activity; (iv) a late lipid accumulation phase, characterized by the rerouting of carbon fluxes within cells. This study explores the potential of Y. lipolytica as an alternative oil producer, by identifying, at the transcriptomic level, the genes potentially involved in the metabolism of oleaginous species.

An approach to the study of gene expression in hepatocarcinogenesis initiation.

In carcinogenesis, determination of gene and protein expression profiles is important for prevention and treatment. Caffeic acid phenethyl ester (CAPE) in a single dose administered before carcinogenic initiation induced by diethylnitrosamine (DEN) prevents the appearance of preneoplastic lesions. On the basis of this approach, the main purpose of this work was to compare the gene expression profiles induced by DEN or a previously administered single dose of CAPE. Using a modified hepatocarcinogenesis-resistant hepatocyte model, male Fischer-344 rats were administered with one intraperitoneal dose of CAPE (20 mg/kg) 12 hours before DEN administration (200 mg/kg). Livers were removed and processed for microarray analysis and reverse transcription-polymerase chain reaction 12 hours after CAPE dosing and 24 hours after DEN administration with or without CAPE. CAPE alone did not alter the expression profile. DEN treatment modified the expression of 665 genes, and CAPE plus DEN induced changes in 1371 genes. DEN treatment increased the expression of genes associated with oxidative stress such as glutathione reductase, genes involved in cell cycle regulation including p53, and modified cytochrome P450. CAPE plus DEN diminished the expression of cytochrome involved in DEN bioactivation such as CYP2B1 as well as the expression of regulators of oxidative stress such as glutathione reductase, GST-kappa and GST-theta, and cell cycle regulators such as p53. Using CAPE as a tool, we uncovered new approaches for studying the altered expression of reactive genes and identifying proteins that will help to propose well-sustained and concrete hypothesis of DEN mechanism of hepatocarcinogenesis initiation.

Celecoxib activates Stat5 and restores or increases the expression of growth hormone-regulated genes in hepatocarcinogenesis.

We have previously evaluated the chemopreventive effect of celecoxib on preneoplastic lesions in rat liver. However, though the effects of celecoxib have been tested in a variety of carcinomas, there has not been a study on the modulation of gene expression in response to this drug. Here, we evaluated the effect of celecoxib on the gene expression profile associated with hepatocarcinogenesis. Male Sprague-Dawley rats underwent the modified resistant hepatocyte model and were fed a diet containing 1500 ppm of celecoxib. Gene expression profiles were evaluated using DNA microarrays and further validations were performed using quantitative PCR, western blotting and immunohistochemical staining. Celecoxib modulated the expression of 46 genes, and those regulated by growth hormone were selected for further analysis. Celecoxib significantly upregulated the expression of the Cyp2b1/2, Cyp3a1, and alpha2-urinary globulin (alpha2uG) genes and restored the expression of Cyp2b3 to normal. The protein expression of Cyp2b1/2 was increased, but the expressions of Cyp3a1 and alpha2uG were only restored to normal levels. The increased Cyp2b1/2 expression in response to celecoxib was mainly confined to preneoplastic lesions. A search for the upstream mediator of these genetic alterations found that carcinogenesis inactivated by 87% the signal transducer and activator of transcription 5 (Stat5), a transcription factor that is activated by growth hormone signaling, but celecoxib treatment restored its activation. In conclusion, these results suggest that celecoxib exerts anticancer effects on altered hepatic cells by restoring mRNA and the protein expression levels of specific genes, in part through the reactivation of Stat5.

Comparative transcriptome analysis between original and evolved recombinant lactose-consuming Saccharomyces cerevisiae strains.

The engineering of Saccharomyces cerevisiae strains for lactose utilization has been attempted with the intent of developing high productivity processes for alcoholic fermentation of cheese whey. A recombinant S. cerevisiae flocculent strain that efficiently ferments lactose to ethanol was previously obtained by evolutionary engineering of an original recombinant that displayed poor lactose fermentation performance. We compared the transcriptomes of the original and the evolved recombinant strains growing in lactose, using cDNA microarrays. Microarray data revealed 173 genes whose expression levels differed more than 1.5-fold. About half of these genes were related to RNA-mediated transposition. We also found genes involved in DNA repair and recombination mechanisms, response to stress, chromatin remodeling, cell cycle control, mitosis regulation, glycolysis and alcoholic fermentation. These transcriptomic data are in agreement with some of the previously identified physiological and molecular differences between the recombinants, and point to further hypotheses to explain those differences.

Proteasome activity deregulation in LEC rat hepatitis: following the insights of transcriptomic analysis.

LEC rats show spontaneous hepatitis and hepatocarcinoma development related to oxidative stress due to abnormal copper accumulation in the liver. We used DNA microarrays bearing 22,012 genes to investigate at the transcriptomic level the progression of the hepatitis in LEC rats in comparison to a control obtained from LEC rats treated with D-penicillamine, a copper chelating agent known to block hepatitis development. Multivariate statistical analyses as partial least square (PLS) regression between transcriptomic data and hepatitis markers in plasma led us to select 483 genes related to hepatitis development in these rats. After a complementary discriminant analysis (PLS-DA), 239 important genes for the separation between the different rat groups were selected. Gene ontology classification revealed an overrepresentation of genes involved in protein metabolism-related functions. More importantly, some genes implicated in proteasome pathway were upregulated. However, analysis of 20S proteasome activity showed that trypsin-like and peptidylglutamyl peptide hydrolase activities were diminished during hepatitis. Because oxidative stress is known to promote the inactivation of the proteasome complex, we propose the deregulation of the proteasome genes expression as a result of oxidative inactivation of proteasome activity during hepatitis in LEC rats. These results bring new insights in the hepatitis and the hepatocarcinogenesis development.

Gene expression profile related to the progression of preneoplastic nodules toward hepatocellular carcinoma in rats.

In this study, we investigated the time course gene expression profile of preneoplastic nodules and hepatocellular carcinomas (HCC) to define the genes implicated in cancer progression in a resistant hepatocyte model. Tissues that included early nodules (1 month, ENT-1), persistent nodules (5 months, ENT-5), dissected HCC (12 months), and normal livers (NL) from adult rats were analyzed by cDNA arrays including 1185 rat genes. Differential genes were derived in each type of sample (n = 3) by statistical analysis. The relationship between samples was described in a Venn diagram for 290 genes. From these, 72 genes were shared between tissues with nodules and HCC. In addition, 35 genes with statistical significance only in HCC and with extreme ratios were identified. Differential expression of 11 genes was confirmed by comparative reverse transcription-polymerase chain reaction, whereas that of 2 genes was confirmed by immunohistochemistry. Members involved in cytochrome P450 and second-phase metabolism were downregulated, whereas genes involved in glutathione metabolism were upregulated, implicating a possible role of glutathione and oxidative regulation. We provide a gene expression profile related to the progression of nodules into HCC, which contributes to the understanding of liver cancer development and offers the prospect for chemoprevention strategies or early treatment of HCC.

Microstructured liposome array.

Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.

Porcine pancreatic alpha-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (alpha-AI1) and structural changes in the alpha-amylase inhibitor complex.

Porcine pancreatic alpha-amylase (PPA) is inhibited by the red kidney bean (Phaseolus vulgaris) inhibitor alpha-AI1 [Eur. J. Biochem. 265 (1999) 20]. Inhibition kinetics were carried out using DP 4900-amylose and maltopentaose as substrate. As shown by graphical and statistical analysis of the kinetic data, the inhibitory mode is of the mixed noncompetitive type whatever the substrate thus involving the EI, EI2, ESI and ESI2 complexes. This contrast with the E2I complex obtained in the crystal and with biophysical studies. Such difference very likely depends on the [I]/[E] ratio. At low ratio, the E2I complex is favoured; at high ratio the EI, ESI and EI2 complexes are formed. The inhibition model also differs from those previously proposed for acarbose [Eur. J. Biochem. 241 (1996) 787 and Eur. J. Biochem. 252 (1998) 100]. In particular, with alpha-AI1, the inhibition takes place only when PPA and alpha-AI are preincubated together before adding the substrate. This indicates that the abortive PPA-alphaAI1 complex is formed during the preincubation period. One additional carbohydrate binding site is also demonstrated yielding the ESI complex. Also, a second protein binding site is found in EI2 and ESI2 abortive complexes. Conformational changes undergone by PPA upon alpha-AI1 binding are shown by higher sensitivity to subtilisin attack. From X-ray analysis of the alpha-AI1-PPA complex (E2I), the major interaction occurs with two hairpin loops L1 (residues 29-46) and L2 (residues 171-189) of alpha-AI1 protruding into the V-shaped active site of PPA. The hydrolysis of alpha-AI1 that accounts for the inhibitory activity is reported.

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis.

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive approximately 100 A layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 micro M with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.