Voluntary medical male circumcision: translating research into the rapid expansion of services in Kenya, 2008-2011

Since the World Health Organization and the Joint United Nations Programme on HIV/AIDS recommended implementation of medical male circumcision (MC) as part of HIV prevention in areas with low MC and high HIV prevalence rates in 2007, the government of Kenya has developed a strategy to circumcise 80% of uncircumcised men within five years. To facilitate the quick translation of research to practice, a national MC task force was formed in 2007, a medical MC policy was implemented in early 2008, and Nyanza Province, the region with the highest HIV burden and low rates of circumcision, was prioritized for services under the direction of a provincial voluntary medical male circumcision (VMMC) task force. The government's early and continuous engagement with community leaders/elders, politicians, youth, and women's groups has led to the rapid endorsement and acceptance of VMMC. In addition, several innovative approaches have helped to optimize VMMC scale-up. Since October 2008, the Kenyan VMMC program has circumcised approximately 290,000 men, mainly in Nyanza Province, an accomplishment made possible through a combination of governmental leadership, a documented implementation strategy, and the adoption of appropriate and innovative approaches. Kenya's success provides a model for others planning VMMC scale-up programs.     

The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth

Metabolism must be coordinated with development to provide the appropriate energetic needs for each stage in the life cycle. Little is known, however, about how this temporal control is achieved. Here, we show that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. dERR mutants die as larvae with low ATP levels and elevated levels of circulating sugars. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells, supporting the dramatic growth that occurs during larval development. This study shows that dERR plays a central role in carbohydrate metabolism, demonstrates that a proliferative metabolic program is used in normal developmental growth, and provides a molecular context to understand the close association between mammalian ERR family members and cancer.     

A targeted proteomics-based pipeline for verification of biomarkers in plasma

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.     

A high-throughput absorbance-based assay for methionine produced by methionine aminopeptidase using S-adenosyl-L-methionine synthetase

Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.     

A genomic map enriched for markers linked to Avr1 in Cronartium quercuum f.sp. fusiforme

A novel approach is presented to map avirulence gene Avr1 in the basidiomycete Cronartium quercuum f.sp. fusiforme, the causal agent of fusiform rust disease in pines. DNA markers tightly linked to resistance gene Fr1 in loblolly pine tree 10-5 were used to classify 10-5 seedling progeny as either resistant or susceptible. A single dikaryotic isolate (P2) heterozygous at the corresponding Avr1 gene was developed by crossing Fr1 avirulent isolate SC20-21 with Fr1 virulent isolate NC2-40. Bulk basidiospore inoculum derived from isolate P2 was used to challenge the pine progeny. The ability to unambiguously marker classify 10-5 progeny as resistant (selecting for virulence) or susceptible (non-selecting) permitted the genetic mapping of the corresponding Avr1 gene by bulked segregant analysis. Using this approach, 14 genetic markers significantly linked to Avr1 were identified and placed within the context of a genome-wide linkage map produced for isolate P2 using samples from susceptible seedlings.     

mTOR drives its own activation via SCF(βTrCP)-dependent degradation of the mTOR inhibitor DEPTOR

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF(βTrCP). DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.     

Evidence for the presence of a critical disulfide bond in the mouse EP3γ receptor

To determine the contribution of cysteines to the function of the mouse E-prostanoid subtype 3 gamma (mEP3γ), we tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from HEK293 cells transfected with mEP3γ C107A or C184A had no detectable radioligand binding. Other mutant mEP3γ receptors had radioligand affinities and receptor densities similar to wild-type. Cell-surface ELISA against the N-terminal HA-tag of C107A and C184A demonstrated 40% and 47% reductions respectively in receptor protein expression at the cell surface, and no radioligand binding was detected as assessed by intact cell radioligand binding experiments. These data suggest a key role for C107 and C184 in both receptor structure/stability and function and is consistent with the presence of a conserved disulfide bond between C107 and C184 in mouse EP3 that is required for normal receptor expression and function. Our results also indicate that if a second disulfide bond is present in the native receptor it is non-essential for receptor assembly or function.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Aging and factors related to running economy

The purpose of this study was to investigate the relationship that age has on factors affecting running economy (RE) in competitive distance runners. Fifty-one male and female subelite distance runners (Young [Y]: 18-39 years [n = 18]; Master [M]: 40-59 years [n = 22]; and Older [O]: 60-older [n = 11]) were measured for RE, step rate, lactate threshold (LT), VO2max, muscle strength and endurance, flexibility, power, and body composition. An RE test was conducted at 4 different velocities (161, 188, 215, and 241 m·min(-1)), with subjects running for 5 minutes at each velocity. The steady-state VO2max during the last minute of each stage was recorded and plotted vs. speed, and a regression equation was formulated. A 1 × 3 analysis of variance revealed no differences in the slopes of the RE regression lines among age groups (y = 0.1827x - 0.2974; R2 = 0.9511 [Y]; y = 0.1988x - 1.0416; R2 = 0.9697 [M]; y = 0.1727x + 3.0252; R2 = 0.9618 [O]). The VO2max was significantly lower in the O group compared to in the Y and M groups (Y = 64.1 ± 3.2; M = 56.8 ± 2.7; O = 44.4 ± 1.7 mlO2·kg(-1)·min(-1)). The maximal heart rate and velocity @ LT were significantly different among all age groups (Y = 197 ± 4; M = 183 ± 2; O = 170 ± 6 b·min(-1) and Y = 289.7 ± 27.0; M = 251.5 ± 32.9; O = 212.3 ± 24.6 m·min(-1), respectively). The VO2max @ LT was significantly lower in the O group compared to in the Y and M groups (Y = 50.3 ± 2.0; M = 48.8 ± 2.9; O = 34.9 ± 3.2 mlO2·kg(-1)·min(-1)). The O group was significantly lower than in the Y and M groups in flexibility, power, and upper body strength. Multiple regression analyses showed that strength and power were significantly related to running velocity. The results from this cross-sectional analysis suggest that age-related declines in running performance are associated with declines in maximal and submaximal cardiorespiratory variables and declines in strength and power, not because of declines in running economy.