A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings

Background

In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.

Methods

A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.

Results

Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.

Conclusion

We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.     

Involvement of Polyamines with Adventitious Root Development in Stem Cuttings of Mung Bean

Spermine enhances the number of adventitious roots developingon stem cuttings of Phaseolus aureus Roxb. This effect is observedwhen spermine is supplied alone to cuttings or in the presenceof indolebutyric acid (IBA). That concentration most effectivein inducing the rooting response also enhances root growth.Other concentrations tested were without effect on growth. Spermidinedoes not influence root number or growth except at high concentration,when it is inhibitory to number only. Methylglyoxal bis(guanylhydrazone)(MGBG) inhibits rooting and root growth in the presence or absenceof IBA. Treatment of stem-cuttings with IBA leads to enhancedlevels of spermine, spermidine and putrescine in the hypocotylprior to development of any root primordia. MGBG reduces thelevels of spermine and spermidine whilst increasing the levelof putrescine. Furthermore, MGBG prevents the IBA-induced increasein spermine and markedly inhibits that in spermidine. Theseresults are consistent with an essential role for polyaminesand their metabolism in the early events which lead to adventitiousroot development. (Received January 10, 1983; Accepted March 17, 1983)     

Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations

Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells.

     

Detection and Quantification of Male-Specific Fetal DNA in the Serum of Pregnant Cynomolgus Monkeys (Macaca fascicularis)

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175–131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.Abbreviations: C_t, threshold cycleAnalysis of cell-free circulating nucleic acids in human maternal plasma or serum has led to the development of risk-free methods for prenatal genetic diagnosis and the assessment of several fetal and maternal conditions, for example, sex determination for paternally inherited diseases, pregnancy-associated complications, sex-linked disorders for ambiguous genitalia, and embryo tracking.^{1,4,12,14,18,19} Technical challenges associated with detecting fetal DNA arise due to the low concentration of fetal DNA in maternal plasma during pregnancy and the difficulty of differentiating the genetic material of the fetus from that of the mother.^5,13,20 Fetal sex determination using sequences derived from the Y chromosome only is relatively simple and has a reported accuracy rate in humans of approximately 99.0% at 7 wk of gestation and 100% after 20 wk, depending on the protocol and methods used.^3,5,17,20 In other species, researchers have used real-time PCR assays during pregnancy to predict fetal sex from cell-free DNA at an accuracy of 100%.^9,10,11 Cell-free fetal DNA in the maternal circulation represents only 3% to 6% of the total free DNA obtained from plasma throughout pregnancy; however, this percentage is variable between pregnancies.^5,13,20In clinical biomedical research, it is essential to develop animal models for human diseases to reveal their mechanisms.^16,22 Continued progress in surgical intervention and molecular medicine suggests that it may soon be possible to develop potential treatments or even cures for several fetal genetic diseases at an early stage of pregnancy.¹⁵ Fetal developmental research during early pregnancy might be facilitated by using cell-free fetal DNA in the maternal blood rather than other methods, such as serum screening and ultrasonography. Nonhuman primates, especially macaques, are useful model animals for studying fetal development because of the similarity of the reproductive characteristics, placental structure, and developmental events between these animals and humans.^9,10 These developmental similarities highlight the importance of the study of cell-free fetal DNA in nonhuman primates and its usefulness as a marker to obtain genetic information about the fetus.In the current study, we investigated the presence of cell-free fetal DNA in the maternal plasma of cynomolgus monkeys by developing and using a standardized PCR system. To this end, we selected the SRY (sex-determining region Y) gene and DYS14 sequences of the cynomolgus monkey to use as sex-associated markers. The Y chromosome-specific sequences in the single-copy sex determination region of SRY and the multicopy (thus yielding increased sensitivity) sequences of DYS14 in the TSPY (testis-specific protein, Y-linked) gene have had wide clinical use in humans as molecular markers for detecting and quantifying cell-free fetal DNA.^3,7 In addition, TSPY has been used in bovines for detecting cell-free fetal DNA² and in rhesus macaques for long-term evaluation of microchimerism.⁸ Given the reports of fetal sex determination in rhesus macaques^9,10 and sheep¹¹ by analyzing Y chromosome-specific sequences from cell-free DNA, we hypothesized that we could predict the fetal sex of cynomolgus monkeys at different stages of gestation. This information has been extremely useful in optimizing the design of experimental studies in biomedical research and in managing a nonhuman primate breeding colony.¹⁰ Because cynomolgus and rhesus macaques are closely related members of the same genus, the current experiments are similar to a previous study.⁹We developed an efficient 2-color multiplex PCR system to detect and quantify fetal DNA in the maternal serum of cynomolgus monkeys during pregnancy. We used 2 loci on the Y chromosome in a single PCR test to minimize the likelihood of false-positive signals. Here we report the results of detection and analysis of fetal DNA at various weeks of gestation and evaluate our PCR system for its ability to determine fetal sex from pregnant monkeys’ cell-free DNA.     

Nox1-dependent reactive oxygen generation is regulated by Rac1

Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Structure of human endothelin-converting enzyme I complexed with phosphoramidon

Endothelin-converting enzyme I (ECE-1) is a mammalian type II integral membrane zinc-containing endopeptidase. ECE-1 catalyzes the final step in the biosynthesis of endothelins in a rate-limiting fashion, through post-translational conversion of the biologically inactive big endothelins. Endothelin-1 overproduction has been implicated in a heterogeneous list of diseases including systemic and pulmonary hypertension, stroke and asthma, cardiac and renal failure. Therefore, ECE-1 is a prime therapeutic target for the regulation of endothelin-1 production in vivo and there is considerable interest in selective inhibitors of this enzyme. Here, we present the crystal structure of the extracellular domain (residues 90-770) of human ECE-1 (C428S) with the generic metalloprotease inhibitor phosphoramidon determined at 2.38 Å resolution. The structure is closely related to that of human NEP, providing essential information for a detailed understanding of ligand-binding, specificity determinants as well as selectivity criteria. Selective inhibitors of ECE-1s should have beneficial effects for the treatment of diseases in which an overproduction of ETs plays a pathogenic role.