First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

BACKGROUND: Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. METHODS AND FINDINGS: We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. CONCLUSIONS: We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian reg

TANK 40 FINAL SB5 CHEMICAL CHARACTERIZATION RESULTS PRIOR TO NP ADDITION

A sample of Sludge Batch 5 (SB5) was pulled from Tank 40 in order to obtain radionuclide inventory analyses necessary for compliance with the Waste Acceptance Product Specifications (WAPS). This sample was also analyzed for chemical composition including noble metals. Prior to radionuclide inventory analyses, a final sample of the H-canyon Np stream will be added to bound the Np addition anticipated for Tank 40. These analyses along with the WAPS radionuclide analyses will help define the composition of the sludge in Tank 40 that is currently being fed to DWPF as SB5. At the Savannah River National Laboratory (SRNL) the 3-L Tank 40 SB5 sample was transferred from the shipping container into a 4-L high density polyethylene vessel and solids allowed to settle overnight. Supernate was then siphoned off and circulated through the shipping container to complete the transfer of the sample. Following thorough mixing of the 3-L sample, a 239 g sub-sample was removed. This sub-sample was then utilized for all subsequent analytical samples. Eight separate aliquots of the slurry were digested, four with HNO{sub 3}/HCl (aqua regia) in sealed Teflon{reg_sign} vessels and four in Na{sub 2}O{sub 2} (alkali or peroxide fusion) using Zr crucibles. Due to the use of Zr crucibles and Na in the peroxide fusions, Na and Zr cannot be determined from this preparation. Additionally, other alkali metals, such as Li and K that may be contaminants in the Na{sub 2}O{sub 2} are not determined from this preparation. Three Analytical Reference Glass - 14 (ARG-1) standards were digested along with a blank for each preparation. The ARG-1 glass allows for an assessment of the completeness of each digestion. Each aqua regia digestion and blank was diluted to 1:100 mL with deionized water and submitted to Analytical Development (AD) for inductively coupled plasma - atomic emission spectroscopy (ICPAES) analysis, inductively coupled plasma - mass spectrometry (ICP-MS) analysis of masses 81-209 and 230-252, and cold vapor atomic absorption (CV-AA) analysis for Hg. Equivalent dilutions of the peroxide fusion digestions and blank were submitted to AD for ICP-AES analysis. Tank 40 SB5 supernate was collected from a mixed slurry sample in the SRNL Shielded Cells and submitted to AD for ICP-AES. Weighted dilutions of slurry were submitted for ion chromatography (IC), total inorganic carbon/total organic carbon (TIC/TOC), and total base analyses. The following conclusions were drawn from the analytical results reported here: (1) The elemental ratios of the major elements for the SB5 WAPS sample, whose major Tank 51 Qualification sample component underwent Al dissolution, are similar to those measured for the SB4 WAPS sample. (2) The elemental composition of this sample and the analyses conducted here are reasonable and consistent with DWPF batch data measurements in light of DWPF pre-sample concentration and SRAT product heel contributions to the DWPF SRAT receipt analyses. (3) Fifty percent of the sulfur in the SB5 WAPS sample is insoluble, and this represents a significantly larger fraction than that observed in previous sludge batches. (4) The noble metal and Ag concentrations predicted from the measured values for the Tank 51 Confirmation sample and Tank 40 SB4 WAPS sample using a two-thirds Tank 51, one-third Tank 40 heel blend ratio used to arrive at the final SB5 composition, agree with the values for the Tank 40 SB5 WAPS sample measured for this report

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

MAGNOX:BUTEX URANIUM BEARING GLASSES PHYSICAL AND CHEMICAL ANALYSIS DATA PACKAGE

Sellafield Ltd (United Kingdom) has requested technical support from the Savannah River National Laboratory (SRNL) to characterize a series of uranium-bearing, mixed alkali borosilicate glasses [WFO (2010)]. The specific glasses to be characterized are based on different blends of Magnox (WRW17 simulant) and Butex (or HASTs 1 and 2) waste types as well as different incorporation rates (or waste loadings) of each blend. Specific Magnox:Butex blend ratios of interest include: 75:25, 60:40, and 50:50. Each of these waste blend ratios will be mixed with a base glass additive composition targeting waste loadings (WLs) of 25, 28, and 32% which will result in nine different glasses. The nine glasses are to be fabricated and physically characterized to provide Sellafield Ltd with the technical data to evaluate the impacts of various Magnox:Butex blend ratios and WLs on key glass properties of interest. It should be noted that the use of 'acceptable' in the Work for Other (WFO) was linked to the results of a durability test (more specifically the Soxhlet leach test). Other processing (e.g., viscosity ({eta}), liquidus temperature (T{sub L})) or product performance (e.g., Product Consistency Test (PCT) results - in addition to the Soxhlet leach test) property constraints were not identified. For example, a critical hold point in the classification of an 'acceptable glass' prior to processing high-level waste (HLW) through the Defense Waste Processing Facility (DWPF) is an evaluation of specific processing and product performance properties against pre-defined constraints. This process is referred to as Slurry Mix Evaporator (SME) acceptability in which predicted glass properties (based on compositional measurements) are compared to predefined constraints to determine whether the glass is acceptable [Brown and Postles (1995)]. As an example, although the nominal melter temperature at DWPF is 1150 C, there is a T{sub L} constraint (without uncertainties applied) of 1050 C. Any glass which has a T{sub L} predicted value > 1050 C would be classified as unacceptable and the SME product would not be transferred to the melter. As another example, consider durability (as defined by the PCT test) and its related constraints to determine acceptability. If the glass composition yields predicted normalized release values that exceed those associated with the Environmental Assessment (EA) glass (with uncertainties applied) then the glass is deemed unacceptable. The issue of acceptability plays a critical role in high level waste processing but without knowing the pre-defined constraints for the UK HLW system, assessments of acceptability of the glasses to be characterized in this study can not be made. The results of this study will be compared to DWPF constraints to provide a benchmark for determining acceptability. The objective of this task is to provide Sellafield Ltd. with the technical data to evaluate the impacts of various Magnox:Butex blend ratios and WLs on key glass properties of interest. The uranium bearing glasses span a compositional region of interest to Sellafield Ltd. and were physically characterized for key processing and product performance properties as defined in the WFO [WFO (2010)]. One of the specific technical issues (as defined in the WFO) is the potential impact of increasing aluminum concentrations on key properties (in particular viscosity)

CHARACTERIZATION OF THE TANK 18F SAMPLES

The Savannah River National Laboratory (SRNL) was asked by Liquid Waste Operations to characterize Tank 18F closure samples. Tank 18F slurry samples analyzed included the liquid and solid fractions derived from the 'as-received' slurry materials along with the floor scrape bottom Tank 18F wet solids. These samples were taken from Tank 18F in March 2009 and made available to SRNL in the same month. Because of limited amounts of solids observed in Tank 18F samples, the samples from the north quadrants of the tank were combined into one North Tank 18F Hemisphere sample and similarly the south quadrant samples were combined into one South Tank 18F Hemisphere sample. These samples were delivered to the SRNL shielded cell. The Tank 18F samples were analyzed for radiological, chemical and elemental components. Where analytical methods yielded additional contaminants other than those requested by the customer, these results were also reported. The target detection limits for isotopes analyzed were 1E-04 {micro}Ci/g for most radionuclides and customer desired detection values of 1E-05 {micro}Ci/g for I-129, Pa-231, Np-237, and Ra-226. While many of the minimum detection limits, as specified in the technical task request and task technical and quality assurance plans were met for the species characterized for Tank 18F, some were not met due to spectral interferences. In a number of cases, the relatively high levels of radioactive species of the same element or a chemically similar element precluded the ability to measure some isotopes to low levels. SRNL, in conjunction with the plant customer, reviewed all these cases and determined that the impacts were negligible

The Impact of Small Molecule Binding on the Energy Landscape of the Intrinsically Disordered Protein C-Myc

Intrinsically disordered proteins are attractive therapeutic targets owing to their prevalence in several diseases. Yet their lack of well-defined structure renders ligand discovery a challenging task. An intriguing example is provided by the oncoprotein c-Myc, a transcription factor that is over expressed in a broad range of cancers. Transcriptional activity of c-Myc is dependent on heterodimerization with partner protein Max. This protein-protein interaction is disrupted by the small molecule 10058-F4 (1), that binds to monomeric and disordered c-Myc. To rationalize the mechanism of inhibition, structural ensembles for the segment of the c-Myc domain that binds to 1 were computed in the absence and presence of the ligand using classical force fields and explicit solvent metadynamics molecular simulations. The accuracy of the computed structural ensembles was assessed by comparison of predicted and measured NMR chemical shifts. The small molecule 1 was found to perturb the composition of the apo equilibrium ensemble and to bind weakly to multiple distinct c-Myc conformations. Comparison of the apo and holo equilibrium ensembles reveals that the c-Myc conformations binding 1 are already partially formed in the apo ensemble, suggesting that 1 binds to c-Myc through an extended conformational selection mechanism. The present results have important implications for rational ligand design efforts targeting intrinsically disordered proteins

Predictors of Marital Satisfaction in Couples That Include a Physician Partner

It is estimated that the divorce rate for marriages in which at least one party is a physician is 10-20% higher than the general population. Predicting the variables that contribute to marital satisfaction, identifying the skills associated with those behaviors and developing a residency curriculum to teach the skills to resident physicians is the focus of this study. Intimacy, conflict resolution and emotional expressiveness have all been identified as important aspects of marital satisfaction. We will determine how each of these variables contributes to marital satisfaction using multilevel logistic regression. The results will be used to develop a skills based training curriculum for residency to improve the lives and well-being of physicians and their families