Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from () (Bath) and () 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function.

In methylotrophic bacteria, which use one-carbon (C1) compounds as a carbon source, methanol is oxidized by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) enzymes. Methylotrophic genomes generally encode two distinct MDHs, MxaF and XoxF. MxaF is a well-studied, calcium-dependent heterotetrameric enzyme whereas XoxF is a lanthanide-dependent homodimer. Recent studies suggest that XoxFs are likely the functional MDHs in many environments. In methanotrophs, methylotrophs that utilize methane, interactions between particulate methane monooxygenase (pMMO) and MxaF have been detected. To investigate the possibility of interactions between pMMO and XoxF, XoxF was isolated from the methanotroph Methylomicrobium buryatense 5GB1C (5G-XoxF). Purified 5G-XoxF exhibits a specific activity of 0.16 μmol DCPIP reduced min mg. The 1.85 Å resolution crystal structure reveals a La(III) ion in the active site, in contrast to the calcium ion in MxaF. The overall fold is similar to other MDH structures, but 5G-XoxF is a monomer in solution. An interaction between 5G-XoxF and its cognate pMMO was detected by biolayer interferometry, with a K value of 50 ± 17 μM. These results suggest an alternative model of MDH-pMMO association, in which a XoxF monomer may bind to pMMO, and underscore the potential importance of lanthanide-dependent MDHs in biological methane oxidation.

Objectives Previous studies found that racial and ethnic minorities may be less likely than non-Hispanic Whites (Whites) to meet existing Medicare medication therapy management (MTM) eligibility criteria. To address these issues, the Centers for Medicare & Medicaid Services (CMS) proposed alternative Medicare MTM eligibility criteria for 2015. Due to opposition to other Part D reforms proposed simultaneously by various stakeholders, CMS rescinded all proposed reforms. This study was conducted to determine whether non-Hispanic Blacks (Blacks) and Hispanics have lower likelihood of meeting the proposed 2015 Medicare MTM eligibility criteria. Methods This retrospective observational analysis used Medical Expenditure Panel Survey data (2010-2011). The final study sample was comprised of 2,721 Whites (weighted to 37,185,896), 917 Blacks (weighted to 4,665,644), and 538 Hispanics (weighted to 3,532,882). Chi-square tests were used to examine racial and ethnic disparities in meeting proposed 2015 MTM eligibility criteria and each component of proposed 2015 MTM eligibility criteria. In multivariate analysis, a logistic regression model was used to control for population socio-demographic and health-related characteristics. Key Findings Compared to Whites with a proportion of MTM eligibility of 58.82%, the eligible proportion was 57.09% (P=0.20) for Blacks, and 48.97% (P<0.0001) for Hispanics, respectively. According to multivariate logistic regression, odds ratios of meeting MTM eligibility for Blacks and Hispanics compared to Whites were 0.74 (95% Confidence Internal [CI] = 0.62-0.88) and 0.53 (95% CI=0.43-0.67), respectively. Conclusions The proposed 2015 MTM eligibility criteria would not eliminate racial and ethnic disparities in MTM eligibility. Alternative MTM eligibility criteria should be devised.

In this study, we obtained a batch of simple sequence repeats (SSRs) from the transcriptome data of Asian moon scallop Amusium pleuronectes and analyzed the distribution and frequency of these SSRs. A total of 7,315 SSRs were obtained from 159,521 unigenes. Bioinformatics tools were employed to design appropriate primers. A total of 4,038 SSR loci had flanking sequences suitable for polymerase chain reaction primer design. One hundred SSR primers were validated and the rate of successful amplification was 78.0%. Fourteen randomly chosen primer pairs were amplified in Beibu Bay population (BP) and Hainan Baimajing population (HP). The number of alleles at each locus ranged from 2 to 3 in two populations, with mean values of 2.214 and 2.143, respectively. The observed heterozygosity, expected heterozygosity and polymorphism information content of BP were 0.463, 0.646 and 0.281, respectively, while those of HP were 0.309, 0.320 and 0.259, respectively. The developed SSR markers will be helpful for further studies on population genetics, genetic linkage construction and chromosome linkage mapping in the species.

African trypanosomiasis is a fatal vector-borne disease caused by the protozoal parasite Trypanosoma brucei. This parasite encodes both de novo and salvage pathways for pyrimidine nucleotide biosynthesis, but in contrast to other eukaryotes, it lacks a dedicated enzyme to interconvert between cytidine (C) and uridine (U) nucleotide pools. Instead, we previously reported that nucleotide interconversion was mediated by thymidine kinase (TK) and an unknown 5’-nucleotidase enzyme. T. brucei encodes multiple potential 5’-nucelotidases, including one from the histidine-aspartate (HD) family (5’-HDNT) and two from the haloacid dehalogenase (HAD1 and HAD2) superfamily of metalloenzymes, but their relative roles and function in nucleotide metabolism remained unexplored. Herein we report that enzymes from both families catalyze dephosphorylation of pyrimidine monophosphate nucleotides, but only 5’-HDNT exhibited high activity on dCMP and was essential for T. brucei growth, while HAD1 and HAD2 were dispensable. Growth arrest upon loss of 5’-HDNT expression was rescued by ectopic expression of human dCMP deaminase (HsDCTD), which creates an alternate route for conversion between C and U pools. Knockout of 5’-HDNT led to depletion of pyrimidine pathway metabolites, altered ratios of pyrimidine to purine nucleotides, and DNA damage. Surprisingly, intracellular glutamine, the precursor of de novo pyrimidine biosynthesis, was also significantly reduced, and isotope tracing assays identified a deficiency in glutamine uptake, suggesting that the glutamine transporter was downregulated in response to loss of 5’-HDNT. This response would likely exacerbate perturbation of pyrimidine pathway homeostasis, suggesting we have uncovered a potential regulated cell death pathway in T. brucei.