Interactions and Transport of a Bioconjugated Peptide Targeting the Mitomembrane.

The Szeto-Schiller (SS) peptides are a subclass of cell-penetrating peptides that can specifically target mitochondria and mediate conditions caused by mitochondrial dysfunction. In this work, we constructed an iron-chelating SS peptide and studied its interaction with a mitochondrial-mimicking membrane using atomistic molecular dynamics (MD) simulations. We report that the peptide/membrane interaction is thermodynamically favorable, and the localization of the peptide to the membrane is driven by electrostatic interactions between the cationic residues and the anionic phospholipid headgroups. The insertion of the peptide into the membrane is driven by hydrophobic interactions between the aromatic side chains in the peptide and the lipid acyl tails. We also probed the translocation of the peptide across the membrane by applying nonequilibrium steered MD simulations and resolved the translocation pathway, free energy profile, and metastable states. We explored four distinct orientations of the peptide along the translocation pathway and found that one orientation was energetically more favorable than the other orientations. We tested a significantly slower pulling velocity on the most thermodynamically favorable system and compared metastable states during peptide translocation. We found that the peptide can optimize hydrophobic interactions with the membrane by having aromatic side chains interacting with the lipid acyl tails instead of forming π-π interactions with each other. The mechanistic insights emerging from our work will potentially facilitate improved peptide design with enhanced activity.

Prevalence and genetic basis of first-line drug resistance of Mycobacterium tuberculosis in Ca Mau, Vietnam.

Background and objective: Data on the prevalence of anti-tuberculous drug resistance and its association with genetic mutations in Mycobacterium tuberculosis are limited. Our study explores the genomics of tuberculosis in Ca Mau, Vietnam. Methods: Patients ≥15 years in Ca Mau Province, Vietnam, were screened annually for tuberculosis between 2014 and 2017. Isolates underwent drug susceptibility testing (DST) using the breakpoint method. DNA was extracted and whole genome sequencing (WGS) was performed. Results: We identified 365 positive sputum cultures for M. tuberculosis and processed 237 for DST and 265 for WGS. Resistance to isoniazid was present in 19.8% (95% CI 14.7 to 24.9%), rifampicin in 3.5% (1.1 to 5.7%) and ethambutol in 2.5% (0.9 to 5.4%) of isolates. Relevant mutations in rpoB gene were detected in 3.8% (1.8 to 6.8%). katG, inhA or fabG1 mutations were found in 19.6% (15.0 to 24.9%) with KatG being most common at 12.8% (9.1-17.5%). We found 38.4% of isolates were of Beijing lineage, 49.4% East-African-Indian lineage and 8.4% European-American lineage. There were no associations between resistance profiles and clinical features. Conclusion: The high burden of isoniazid resistance and the katG mutation highlights the challenges facing Vietnam in its efforts to achieve its EndTB goals.

Establishment and characterisation of cell lines and xenograft mouse models of canine systemic histiocytosis and disseminated histiocytic sarcoma.

Canine histiocytic proliferative disorders include reactive diseases (histiocytosis) and neoplastic diseases (histiocytic sarcoma [HS]), however discrimination is challenging due to their overlapping pathological features. In the present study, novel cell lines and xenograft mouse models of systemic histiocytosis (SyH) and disseminated HS were established, and examined together with cell lines previously established from localized HS and Langerhans cell histiocytosis (LCH). The chromosomal numbers of the SyH and HS cell lines were abnormal, and their population doubling time and morphological features were comparable. Immunophenotypically, SyH and HS cells were CD204+/E-cadherin+ in vitro and in vivo, like their original tissues. Western blot analysis for E-cadherin revealed an immunopositive band of full-length E-cadherin (120 kDa) in cultured cells of localized HS and LCH but not in disseminated HS and SyH; expression level was weaker in localized HS than in LCH. An immunopositive band of fragmented E-cadherin (45 kDa) was detected in cell lines of disseminated HS and SyH. These results suggest that cultured SyH cells have features that are similar to disseminated HS, including chromosomal aberration, high proliferation activity, weak cell adhesion, and expression of fragmented E-cadherin. Fragmentation of the E-cadherin cell adhesion molecule may be associated with the loss of cell adhesion and increased abilities of invasion and migration of neoplastic cells. The established cell lines and xenograft mouse models will aid in understanding the pathogenesis and developing novel treatments of canine histiocytic proliferative disorders.

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C-S bond cleavage, substrate scope, and an unexpected role for the tetramer.

Bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C-S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD's functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.

Structure and function of the two-component flavin-dependent methanesulfinate monooxygenase within bacterial sulfur assimilation.

Methyl sulfur compounds are a rich source of environmental sulfur for microorganisms, but their use requires redox systems. The bacterial sfn and msu operons contain two-component flavin-dependent monooxygenases for dimethylsulfone (DMSO2) assimilation: SfnG converts DMSO2 to methanesulfinate (MSI-), and MsuD converts methanesulfonate (MS-) to sulfite. However, the enzymatic oxidation of MSI- to MS- has not been demonstrated, and the function of the last enzyme of the msu operon (MsuC) is unresolved. We employed crystallographic and biochemical studies to identify the function of MsuC from Pseudomonas fluorescens. The crystal structure of MsuC adopts the acyl-CoA dehydrogenase fold with putative binding sites for flavin and MSI-, and functional assays of MsuC in the presence of its oxidoreductase MsuE, FMN, and NADH confirm the enzymatic generation of MS-. These studies reveal that MsuC converts MSI- to MS- in sulfite biosynthesis from DMSO2.

Community-wide Screening for Tuberculosis in a High-Prevalence Setting.

BACKGROUND: The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress. METHODS: We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012. RESULTS: In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test for Mycobacterium tuberculosis (prevalence ratio, 0.56; 95% confidence interval [CI], 0.40 to 0.78; P<0.001). The prevalence of tuberculosis infection in children born in 2012 was 3.3% in the intervention group and 2.6% in the control group (prevalence ratio, 1.29; 95% CI, 0.70 to 2.36; P = 0.42). CONCLUSIONS: Three years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).

Serum 25-hydroxyvitamin D but not dietary vitamin D intake is associated with hemoglobin in women of reproductive age in rural northern Vietnam.

BACKGROUND AND OBJECTIVES: Hypovitaminosis D and anemia are both prevalent in Vietnam, and low vitamin D status may be a risk factor for anemia. This study aimed to 1) describe vitamin D intake and its determinants, and 2) examine the associations of vitamin D intake and serum 25(OH)D concentrations with hemoglobin and anemia. METHODS AND STUDY DESIGN: We used data from the baseline survey of a pre-conceptual micronutrient supplementation trial in women of reproductive age (WRA) in Thai Nguyen, Vietnam (N = 4961). Vitamin D intake was estimated using a semi-quantitative food frequency questionnaire (FFQ). Multivariable regression models were used for the analyses. RESULTS: Median vitamin D intake was 0.2 µg/d (8.0 IU) [IQR: 0.4]. Age, being a farmer, food insecurity, and body mass index (BMI) were inversely associated with vitamin D intake, while socioeconomic status (SES), total energy intake, and education were positively associated with vitamin D intake. Vitamin D intake was not associated with hemoglobin concentration or anemia after adjusting for age, BMI, total energy intake, transferrin receptor, C-reactive protein, α1-acid glycoprotein, SES, occupation, education, ethnicity, and food insecurity (P = 0.56 and P = 0.65 for hemoglobin and anemia, respectively). Controlling for the same covariates, 25(OH)D <50 nmol/L (vs. ≥50 nmol/L) was associated with decreased hemoglobin concentrations (ß = -0.91 (SE:0.42), P = 0.03), but not with anemia (P = 0.11). CONCLUSIONS: Low vitamin D status may be linked to reduced hemoglobin concentrations, but the role of diet in this association was not evident in this population of WRA in Vietnam where dietary vitamin D intake was very low.

Impact of Preconception Micronutrient Supplementation on Anemia and Iron Status during Pregnancy and Postpartum: A Randomized Controlled Trial in Rural Vietnam.

OBJECTIVE: Preconception micronutrient interventions may be a promising approach to reduce anemia and iron deficiency during pregnancy, but currently we have limited data to inform policies. We evaluated whether providing additional pre-pregnancy weekly iron-folic acid (IFA) or multiple micronutrient (MM) supplements compared to only folic acid (FA) improves iron status and anemia during pregnancy and early postpartum. METHODS: We conducted a double blind randomized controlled trial in which 5011 Vietnamese women were provided with weekly supplements containing either only 2800 µg FA (control group), IFA (60 mg Fe and 2800 µg FA) or MM (15 micronutrients with similar amounts of IFA). All women who became pregnant (n = 1813) in each of the 3 groups received daily IFA (60 mg Fe and 400 µg FA) through delivery. Hematological indicators were assessed at baseline (pre-pregnancy), during pregnancy, 3 months post-partum, and in cord blood. Adjusted generalized linear models were applied to examine the impact of preconception supplementation on anemia and iron stores, using both intention to treat and per protocol analyses (women consumed supplements ≥ 26 weeks before conception). RESULTS: At baseline, 20% of women were anemic, but only 14% had low iron stores (ferritin <30 µg/L) and 3% had iron deficiency (ferritin <12 µg/L). The groups were balanced for baseline characteristics. Anemia prevalence increased during pregnancy and post-partum but was similar among intervention groups. In intention to treat analyses, prenatal ferritin was significantly higher among women receiving MM (geometric mean (µg/L) [95% CI]: 93.6 [89.3-98.2]) and IFA (91.9 [87.6-96.3]) compared to control (85.3 [81.5-89.2]). In per protocol analyses, women receiving MM or IFA had higher ferritin 3 months postpartum (MM 118.2 [109.3-127.8]), IFA 117.8 [108.7-127.7] vs control 101.5 [94.0-109.7]) and gave birth to infants with greater iron stores (MM 184.3 [176.1-192.9]), IFA 189.9 [181.6-198.3] vs control 175.1 [167.9-182.6]). CONCLUSION: Preconception supplementation with MM or IFA resulted in modest increases in maternal and infant iron stores but did not impact anemia. Further research is needed to characterize the etiology of anemia in this population and identify effective interventions for reducing prenatal anemia. TRIAL REGISTRATION: ClinicalTrials.Gov NCT01665378.

Germline Genetic Variants in the Wnt/ß-Catenin Pathway as Predictors of Colorectal Cancer Risk.

BACKGROUND: The Wnt/ß-catenin signaling pathway plays a key role in stem cell maintenance in the colorectum. Rare high-penetrance genetic mutations in components of this pathway result in familial colorectal cancer, yet the impact of common, germline variants remains unknown. METHODS: We assessed 172 variants in 26 genes from the Wnt/ß-catenin pathway in 809 colorectal cancer cases and 814 healthy controls, followed by replication of the top findings in another 691 cases and 775 controls. In silico informatic tools were used to predict functional effects of variants. RESULTS: Eighteen SNPs in the pathway were significantly associated with colorectal cancer risk (P < 0.05) in the discovery phase. We observed a significant dose-response increase in colorectal cancer risk by number of risk genotypes carried (P = 4.19 × 10(-8)). Gene-based analysis implicated CSNK1D (P = 0.014), FZD3 (P = 0.023), and APC (P = 0.027) as significant for colorectal cancer risk. In the replication phase, FZD3:rs11775139 remained significantly associated with reduced risk with a pooled OR of 0.85 [95% confidence interval (CI), 0.76-0.94, P = 0.001]. Although borderline significant in the replication population, APC:rs2545162 was highly significant in the pooled analysis-OR, 1.42; 95% CI, 1.16-1.74; P = 0.00085. Functional assessment identified several potential biologic mechanisms underlying these associations. CONCLUSIONS: Our findings suggest that common germline variants in the Wnt/ß-catenin pathway may be involved in colorectal cancer development. IMPACT: These variants may be informative in colorectal cancer risk assessment to identify individuals at increased risk who would be candidates for screening.

Genetic variation among world populations: inferences from 100 Alu insertion polymorphisms.

We examine the distribution and structure of human genetic diversity for 710 individuals representing 31 populations from Africa, East Asia, Europe, and India using 100 Alu insertion polymorphisms from all 22 autosomes. Alu diversity is highest in Africans (0.349) and lowest in Europeans (0.297). Alu insertion frequency is lowest in Africans (0.463) and higher in Indians (0.544), E. Asians (0.557), and Europeans (0.559). Large genetic distances are observed among African populations and between African and non-African populations. The root of a neighbor-joining network is located closest to the African populations. These findings are consistent with an African origin of modern humans and with a bottleneck effect in the human populations that left Africa to colonize the rest of the world. Genetic distances among all pairs of populations show a significant product-moment correlation with geographic distances (r = 0.69, P < 0.00001). F(ST), the proportion of genetic diversity attributable to population subdivision is 0.141 for Africans/E. Asians/Europeans, 0.047 for E. Asians/Indians/Europeans, and 0.090 for all 31 populations. Resampling analyses show that approximately 50 Alu polymorphisms are sufficient to obtain accurate and reliable genetic distance estimates. These analyses also demonstrate that markers with higher F(ST) values have greater resolving power and produce more consistent genetic distance estimates.

Non-traditional Alu evolution and primate genomic diversity.

Alu elements belonging to the previously identified "young" subfamilies are thought to have inserted in the human genome after the divergence of humans from non-human primates and therefore should not be present in non-human primate genomes. Polymerase chain reaction (PCR) based screening of over 500 Alu insertion loci resulted in the recovery of a few "young" Alu elements that also resided at orthologous positions in non-human primate genomes. Sequence analysis demonstrated these "young" Alu insertions represented gene conversion events of pre-existing ancient Alu elements or independent parallel insertions of older Alu elements in the same genomic region. The level of gene conversion between Alu elements suggests that it may have a significant influence on the single nucleotide diversity within the genome. All the instances of multiple independent Alu insertions within the same small genomic regions were recovered from the owl monkey genome, indicating a higher Alu amplification rate in owl monkeys relative to many other primates. This study suggests that the majority of Alu insertions in primate genomes are the products of unique evolutionary events.