Molecular Characteristics of Mycobacterium tuberculosis Strains Isolated from Cutaneous Tuberculosis Patients in China.

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.

Molecular insight into the differential anti-androgenic activity of resveratrol and its natural analogs: in silico approach to understand biological actions.

The androgen receptor (AR) is a therapeutic target for the treatment of prostate cancer. Androgen receptor reactivation during the androgen-independent stage of prostate cancer is mediated by numerous mechanisms including expression of AR mutants and splice variants that become non-responsive to conventional anti-androgenic agents. Resveratrol and its natural analogs exhibit varying degrees of anti-androgenic effects on tumor growth suppression in prostate cancer. However, the structural basis for the observed differential activity remains unknown. Here, anti-androgenic activities of resveratrol and its natural analogs, namely, pterostilbene, piceatannol and trimethoxy-resveratrol were studied in LNCaP cells expressing T877A mutant AR and atomistic simulations were employed to establish the structure activity relationship. Interestingly, essential hydrogen bonding contacts and the binding energies of resveratrol analogs with AR ligand binding domain (LBD), emerge as key differentiating factors for varying anti-androgenic action. Among all the analogs, pterostilbene exhibited strongest anti-androgenic activity and its binding energy and hydrogen bonding interactions pattern closely resembled pure anti-androgen, flutamide. Principal component analysis of our simulation studies revealed that androgenic compounds bind more strongly to AR LBD compared to anti-androgenic compounds and provide conformational stabilization of the receptor in essential subspace. The present study provides critical insight into the structure-activity relationship of the anti-androgenic action of resveratrol analogs, which can be translated further to design novel highly potent anti-androgenic stilbenes.

Dietary pterostilbene is a novel MTA1-targeted chemopreventive and therapeutic agent in prostate cancer.

Overexpression of the epigenetic modifier metastasis-associated protein 1 (MTA1) is associated with aggressive human prostate cancer. The purpose of this study was to determine MTA1- targeted chemopreventive and therapeutic efficacy of pterostilbene, a natural potent analog of resveratrol, in pre-clinical models of prostate cancer. Here, we show that high levels of MTA1 expression in Pten-loss prostate cooperate with key oncogenes, including c-Myc and Akt among others, to promote prostate cancer progression. Loss-of-function studies using human prostate cancer cells indicated direct involvement of MTA1 in inducing inflammation and epithelial-to-mesenchymal transition. Importantly, pharmacological inhibition of MTA1 by pterostilbene resulted in decreased proliferation and angiogenesis and increased apoptosis. This restrained prostatic intraepithelial neoplasia (PIN) formation in prostate-specific Pten heterozygous mice and reduced tumor development and progression in prostate-specific Pten-null mice. Our findings highlight MTA1 as a key upstream regulator of prostate tumorigenesis and cancer progression. More significantly, it offers pre-clinical proof for pterostilbene as a promising lead natural agent for MTA1-targeted chemopreventive and therapeutic strategy to curb prostate cancer.

[Longitudinal study on infantile nocturnal sleep-wake pattern developmental trajectory with Actiwatch].

OBJECTIVE: To understand the infantile nocturnal sleep-wake pattern developmental trajectory with Actiwatch, which would benefit the clinical assessment of infantile sleep. METHOD: This study was a longitudinal study conducted between 7 Oct, 2009-30 Oct, 2011 in 10 hospitals of 9 cities of China ( Beijing, Xi'an, Qingdao, Wuhan, Changsha, Chongqing, Huzhou, Xiamen and Liuzhou). Actiwatch was used to track the sleep-wake pattern development trajectory of healthy infants in the first year of life in the home setting. Participating infants were followed up at 10th day and 28th day during the first month, and then monthly from the second to the sixth month after birth, and then at ninth and twelve months of age respectively. Meanwhile, infantile sleep was observed continuously for about 60 hours at each visit. According to the characteristics of repeated measurement data of this study, two-level random effect model was adopted to analyze the trend of infantile nocturnal sleep-wake parameters changing with age, and the gender difference. RESULT: A total of 473 healthy infants were included in this study, among whom 246 (52.0%) were boys, and 227 (48.0%) were girls; 355 (75.1%) infants completed the whole year follow-up survey. With infants' age increasing, the latency of infants' nighttime sleep onset decreased from 66.8 minutes on 10th day to 15.5-18.7 minutes at 6-12 months of age. The number of night wakes also decreased with age, while uninterrupted sleep periods lengthened with age. On the 10th day, there were 3.0 times of nightwaking on average, and the longest continuous sleeping interval lasted for 227.6 minutes on average. At 12-month of age, infants could sleep continuously for 350.9 minutes at most on average, while the number of nightwaking decreased to 1.6 times per night on average. Generally, nighttime sleep efficiency increased from 66.3% on the 10th day to 86.3% at 12-month of age. The differences of sleep-wake patterns between boys and girls presented as boys' nocturnal longest uninterrupted sleep period was 19 minutes shorter(266.6 vs. 285.6 min), and the average nighttime sleep efficiency was 2.2% lower (74.2% vs. 76.4%) compared with girls respectively. And the differences of sleep efficiency between boys and girls reduced gradually along with the growth. CONCLUSION: During the first 6 months after birth, infantile sleep-wake pattern undergo obvious change. The capability of sleep-onset and uninterrupted sleep improved with age, and the sleep efficiency increased.

Differing lifestyles of Staphylococcus epidermidis as revealed through Bayesian clustering of multilocus sequence types.

Staphylococcus epidermidis is part of the normal bacterial flora of human skin and a leading cause of infections associated with indwelling medical devices. Previous phylogenetic analyses of subgenomic data have been unable to distinguish between S. epidermidis strains with nosocomial or commensal lifestyles, despite the identification of specific phenotypes and accessory genes that may contribute to such lifestyles. To attempt to better define the population structure of this species, the international S. epidermidis multilocus sequence typing database was analyzed with the Bayesian clustering programs STRUCTURE and BAPS. A total of six genetic clusters (GCs) were identified. A local population of S. epidermidis from clinical specimens was classified according to these six GCs, and further characterized for antibiotic susceptibilities, biofilm, and various genetic markers. GC5 was abundant and significantly enriched for isolates that were resistant to four classes of antibiotics, high biofilm production, and positive for the virulence markers icaA, IS256, and sesD/bhp, indicating its potential clinical relevance. In contrast, GC2 was rare and contained the only isolates positive for the putative commensal marker, fdh. GC1 and GC6 were abundant but not significantly associated with any of the examined characteristics, except for sesF/aap and GC6. GC3 was rare and identified as a potential genetic sink that received, but did not donate, core genetic material from other GCs. In conclusion, population genetics analyses were essential for identifying clusters of strains that may differ in their adaptation to nosocomial or commensal lifestyles. These results provide a new, population genetics framework for studying S. epidermidis.

Nuclear MTA1 overexpression is associated with aggressive prostate cancer, recurrence and metastasis in African Americans.

Metastasis-associated protein 1 (MTA1), a negative epigenetic modifier, plays a critical role in prostate cancer (PCa) progression. We hypothesized that MTA1 overexpression in primary tumor tissues can predict PCa aggressiveness and metastasis. Immunohistochemical staining of MTA1 was done on archival PCa specimens from University of Mississippi Medical Center and University of Iowa. We found that nuclear MTA1 overexpression was positively correlated with the severity of disease progression reaching its highest levels in metastatic PCa. Nuclear MTA1 overexpression was significantly associated with Gleason > 7 tumors in African Americans but not in Caucasians. It was also a predictor of recurrent disease. We concluded that MTA1 nuclear overexpression may be a prognostic indicator and a future therapeutic target for aggressive PCa in African American men. Our findings may be useful for categorizing African American patients with a higher probability of recurrent disease and metastasis from those who are likely to remain metastasis-free.

Multilocus sequence typing and further genetic characterization of the enigmatic pathogen, Staphylococcus hominis.

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.

Molecular signatures identify a candidate target of balancing selection in an arcD-like gene of Staphylococcus epidermidis.

A comparative population genetics study revealed high levels of nucleotide polymorphism and intermediate-frequency alleles in an arcC gene of Staphylococcus epidermidis, but not in a homologous gene of the more aggressive human pathogen, Staphylococcus aureus. Further investigation showed that the arcC genes used in the multilocus sequence typing schemes of these two species were paralogs. Phylogenetic analyses of arcC-containing loci, including the arginine catabolic mobile element, from both species, suggested that these loci had an eventful history involving gene duplications, rearrangements, deletions, and horizontal transfers. The peak signatures in the polymorphic S. epidermidis locus were traced to an arcD-like gene adjacent to arcC; these signatures consisted of unusually elevated Tajima's D and π/K ratios, which were robust to assumptions about recombination and species divergence time and among the most elevated in the S. epidermidis genome. Amino acid polymorphisms, including one that differed in polarity and hydropathy, were located in the peak signatures and defined two allelic lineages. Recombination events were detected between these allelic lineages and potential donors and recipients of S. epidermidis were identified in each case. By comparison, the orthologous gene of S. aureus showed no unusual signatures. The ArcD-like protein belonged to the unknown ion transporter 3 family and appeared to be unrelated to ArcD from the arginine deiminase pathway. These studies report the first comparative population genetics results for staphylococci and the first statistical evidence for a candidate target of balancing selection in S. epidermidis.

Methicillin-resistant Staphylococcus aureus sequence type 239-III, Ohio, USA, 2007-2009.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen that has diverse molecular heterogeneity. Most MRSA strains in the United States are pulsed-field gel electrophoresis USA100 sequence type (ST) 5 and USA300 ST8. Infections with MRSA ST239-III are common and found during health care-associated outbreaks. However, this strain has been rarely reported in the United States. As part of a study supported by the Prevention Epicenter Program of the Centers for Disease Control and Prevention (Atlanta, GA, USA), which evaluated transmission of MRSA among hospitals in Ohio, molecular typing identified 78 (6%) of 1,286 patients with MRSA ST239-III infections. Ninety-five percent (74/78) of these infections were health care associated, and 65% (51/78) of patients had histories of invasive device use. The crude case-fatality rate was 22% (17/78). Identification of these strains, which belong to a virulent clonal group, emphasizes the need for molecular surveillance.

Diversity of potential short tandem repeats in Mycobacterium leprae and application for molecular typing.

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multi-case households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.

Diversity of potential short tandem repeats in Mycobacterium leprae and application for molecular typing

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multi-case households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.

Polymorphism in the rpoT gene in Mycobacterium leprae isolates obtained from Latin American countries and its possible correlation with the spread of leprosy.

The genotypes of Mycobacterium leprae isolates originating from Mexico, Peru and Paraguay were analysed for the polymorphism of short tandem repeats in the rpoT gene. The genotype with four copies of the six-base tandem repeats in the rpoT gene was prominently predominant in Mexico, but the genotype of all isolates from Peru and Paraguay contained three copies of the six-base tandem repeats. These obvious different distributions might reflect the spread of leprosy by the different strains of M. leprae harboured by the various human races that moved to the American continent, as has been demonstrated in other infectious diseases.

[Molecular epidemiology of the leprosy].

Application of molecular biological techniques to the epidemiological study of leprosy is described. Studies of detecting Mycobacterium leprae DNA in samples of the nasal mucus are discussed in terms of the epidemiology and the significance of high prevalence. Epidemiological studies on the transmission of leprosy and correlation between geographic distribution of different M. leprae rpoT genotypes and prehistoric spread of the leprosy by genotyping based on the genomic polymorphism are introduced.

Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission.

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.

A mutation at codon 516 in the rpoB gene of Mycobacterium leprae confers resistance to rifampin.

A missense mutation at codon 516 in the rpoB gene of Mycobacterium leprae conferring rifampin resistance was confirmed by the correlation between sequencing results and mouse footpad assay. The isolate was obtained from a relapsed lepromatous leprosy patient. This is the first report on the complete concordance between the mutation located at codon 516 in the rpoB gene and the corresponding resistance to rifampin in leprosy. The novel profile of mutation in the rpoB gene will contribute to the comprehensive understanding of rifampin resistant patterns and offer a useful tool for developing simple and rapid drug susceptibility testing approaches, which would promise more effective and successful control of leprosy.

Leprosy in China.

Leprosy has definitely been present in China for at least 2,000 years. Through painstaking efforts over the past half century, China has put leprosy under control and reached the WHO's target at elimination of leprosy at the national and subnational level. But difficulties as well as problems like disabilities, discrimination, drug-resistance and dismissing of research still remain in the control of leprosy. Highly attention should be continuously paid on to attain the prevalence rate of less than 0.1/10,000 and the incidence rate below 0.5/100,000 in all counties (cities) throughout the country by the year 2010.

A study on a possibility of predicting early relapse in leprosy using a ND-O-BSA based ELISA.

Serological methods have been used for detecting infection with Mycobacterium leprae. We have applied a serological test to explore the possibility it could detect a bacterial relapse among patients who have been cured with chemotherapy. More specifically we used an indirect enzyme-linked immunosorbant assay (ELISA) using the natural disaccharide (ND) of the phenolic glycolipid antigen of M. leprae linked to bovine serum albumin as antigen. Antibody levels were measured in sera from normal controls, active leprosy cases, cured leprosy patients, and relapsing leprosy patients. We correlated antibody levels with the type of leprosy, the bacterial index, and with relapse among cured leprosy patients. In our hands, the ND-ELISA, when applied to screening for infection with M. leprae, had excellent sensitivity, specificity, positive and negative predictive values, and both a low false positive rate and a low false negative rate. Antibody levels gradually increased among active patients from the tuberculoid to the lepromatous end of the leprosy spectrum. There was a year-by-year fall in antibody levels in patients responding to chemotherapy. Antibody levels and the bacterial index were correlated using the Spearman's rank correlation method. Serial antibody levels were measured in 666 leprosy patients after being cured with dapsone monotherapy. Over a three year follow up, 95 multibacillary patients became antibody positive and 12 of them had bacterial relapses of their disease. In contrast, among 335 cases that remained antibody negative, only one relapse was seen. Among 44 paucibacillary cured patients who became antibody positive, there was one relapse. There were 192 such patients who remained antibody negative and one relapsed. The risk of relapse is 6.7 times higher among cured multibacillary patients compared to cured paucibacillary patients. Overall, the cumulative relapse rate among antibody positive cases was 13.7%, compared to 0.4% among antibody negative patients. We conclude that the ND-ELISA is a useful tool both for screening for early infection with M. leprae and for predicting a relapse in cured patients, particularly in cured multibacillary patients.

A study on a possibility of predicting early relapse in leprosy using a ND-O-BSA based ELISA

Serological methods have been used for detecting infection with Mycobacterium leprae. We have applied a serological test to explore the possibility it could detect a bacterial relapse among patients who have been cured with chemotherapy. More specifically we used an indirect enzyme-linked immunosorbant assay (ELISA) using the natural disaccharide (ND) of the phenolic glycolipid antigen of M. leprae linked to bovine serum albumin as antigen. Antibody levels were measured in sera from normal controls, active leprosy cases, cured leprosy patients, and relapsing leprosy patients. We correlated antibody levels with the type of leprosy, the bacterial index, and with relapse among cured leprosy patients. In our hands, the ND-ELISA, when applied to screening for infection with M. leprae, had excellent sensitivity, specificity, positive and negative predictive values, and both a low false positive rate and a low false negative rate. Antibody levels gradually increased among active patients from the tuberculoid to the lepromatous end of the leprosy spectrum. There was a year-by-year fall in antibody levels in patients responding to chemotherapy. Antibody levels and the bacterial index were correlated using the Spearman's rank correlation method. Serial antibody levels were measured in 666 leprosy patients after being cured with dapsone monotherapy. Over a three year follow up, 95 multibacillary patients became antibody positive and 12 of them had bacterial relapses of their disease. In contrast, among 335 cases that remained antibody negative, only one relapse was seen. Among 44 paucibacillary cured patients who became antibody positive, there was one relapse. There were 192 such patients who remained antibody negative and one relapsed. The risk of relapse is 6.7 times higher among cured multibacillary patients compared to cured paucibacillary patients. Overall, the cumulative relapse rate among antibody positive cases was 13.7%, compared to 0.4% among antibody negative patients. We conclude that the ND-ELISA is a useful tool both for screening for early infection with M. leprae and for predicting a relapse in cured patients, particularly in cured multibacillary patients.