A Novel Cholinergic Action of Alcohol and the Development of Tolerance to That Effect in Caenorhabditis elegans

The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102–149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.     

The safety of adult male circumcision in HIV-infected and uninfected men in Rakai, Uganda

Background

The objective of the study was to compare rates of adverse events (AEs) related to male circumcision (MC) in HIV-positive and HIV-negative men in order to provide guidance for MC programs that may provide services to HIV-infected and uninfected men.

Methods and Findings

A total of 2,326 HIV-negative and 420 HIV-positive men (World Health Organization [WHO] stage I or II and CD4 counts > 350 cells/mm³) were circumcised in two separate but procedurally identical trials of MC for HIV and/or sexually transmitted infection prevention in rural Rakai, Uganda. Participants were followed at 1–2 d and 5–9 d, and at 4–6 wk, to assess surgery-related AEs, wound healing, and resumption of intercourse. AE risks and wound healing were compared in HIV-positive and HIV-negative men. Adjusted odds ratios (AdjORs) were estimated by multiple logistic regression, adjusting for baseline characteristics and postoperative resumption of sex. At enrollment, HIV-positive men were older, more likely to be married, reported more sexual partners, less condom use, and higher rates of sexually transmitted disease symptoms than HIV-negative men. Risks of moderate or severe AEs were 3.1/100 and 3.5/100 in HIV-positive and HIV-negative participants, respectively (AdjOR 0.91, 95% confidence interval [CI] 0.47–1.74). Infections were the most common AEs (2.6/100 in HIV-positive versus 3.0/100 in HIV-negative men). Risks of other complications were similar in the two groups. The proportion with completed healing by 6 wk postsurgery was 92.7% in HIV-positive men and 95.8% in HIV-negative men (p = 0.007). AEs were more common in men who resumed intercourse before wound healing compared to those who waited (AdjOR 1.56, 95% CI 1.05–2.33).

Conclusions

Overall, the safety of MC was comparable in asymptomatic HIV-positive and HIV-negative men, although healing was somewhat slower among the HIV infected. All men should be strongly counseled to refrain from intercourse until full wound healing is achieved.Trial registration: http://www.ClinicalTrials.gov; for HIV-negative men, #{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073 and for HIV-positive men, #{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073.     

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication.     

Neuronal Development in Caenorhabditis elegans Is Regulated by Inhibition of an MLK MAP Kinase Pathway

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cell-autonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway.     

Effect of a brief video intervention on incident infection among patients attending sexually transmitted disease clinics

Background

Sexually transmitted disease (STD) prevention remains a public health priority. Simple, practical interventions to reduce STD incidence that can be easily and inexpensively administered in high-volume clinical settings are needed. We evaluated whether a brief video, which contained STD prevention messages targeted to all patients in the waiting room, reduced acquisition of new infections after that clinic visit.

Methods and Findings

In a controlled trial among patients attending three publicly funded STD clinics (one in each of three US cities) from December 2003 to August 2005, all patients (n = 38,635) were systematically assigned to either a theory-based 23-min video depicting couples overcoming barriers to safer sexual behaviors, or the standard waiting room environment. Condition assignment alternated every 4 wk and was determined by which condition (intervention or control) was in place in the clinic waiting room during the patient''s first visit within the study period. An intent-to-treat analysis was used to compare STD incidence between intervention and control patients. The primary endpoint was time to diagnosis of incident laboratory-confirmed infections (gonorrhea, chlamydia, trichomoniasis, syphilis, and HIV), as identified through review of medical records and county STD surveillance registries. During 14.8 mo (average) of follow-up, 2,042 patients (5.3%) were diagnosed with incident STD (4.9%, intervention condition; 5.7%, control condition). In survival analysis, patients assigned to the intervention condition had significantly fewer STDs compared with the control condition (hazard ratio [HR], 0.91; 95% confidence interval [CI], 0.84 to 0.99).

Conclusions

Showing a brief video in STD clinic waiting rooms reduced new infections nearly 10% overall in three clinics. This simple, low-intensity intervention may be appropriate for adoption by clinics that serve similar patient populations.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00137670","term_id":"NCT00137670"}}NCT00137670).     

The Association between Sulfonylurea Use and All-Cause and Cardiovascular Mortality: A Meta-Analysis with Trial Sequential Analysis of Randomized Clinical Trials

BackgroundSulfonylureas are an effective and inexpensive treatment for type 2 diabetes. There is conflicting data about the safety of these drugs regarding mortality and cardiovascular outcomes. The objective of the present study was to evaluate the safety of the sulfonylureas most frequently used and to use trial sequential analysis (TSA) to analyze whether the available sample was powered enough to support the results.ConclusionsSulfonylureas are not associated with increased risk for all-cause mortality, cardiovascular mortality, myocardial infarction, or stroke. Current evidence supports the safety of sulfonylureas; an absolute risk of 0.5% could be firmly discarded.

Review registration

PROSPERO CRD42014004330     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Efficacy of Pneumococcal Nontypable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV) in Young Latin American Children: A Double-Blind Randomized Controlled Trial

BackgroundThe relationship between pneumococcal conjugate vaccine–induced antibody responses and protection against community-acquired pneumonia (CAP) and acute otitis media (AOM) is unclear. This study assessed the impact of the ten-valent pneumococcal nontypable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) on these end points. The primary objective was to demonstrate vaccine efficacy (VE) in a per-protocol analysis against likely bacterial CAP (B-CAP: radiologically confirmed CAP with alveolar consolidation/pleural effusion on chest X-ray, or non-alveolar infiltrates and C-reactive protein ≥ 40 µg/ml); other protocol-specified outcomes were also assessed.ConclusionsEfficacy was demonstrated against a broad range of pneumococcal diseases commonly encountered in young children in clinical practice.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00466947","term_id":"NCT00466947"}}NCT00466947Please see later in the article for the Editors'' Summary     

Addition of Lidocaine Injection Immediately before Physiotherapy for Frozen Shoulder: A Randomized Controlled Trial

The intraarticular injection of lidocaine immediately before a physiotherapy session may relieve pain during the stretching and mobilization of the affected joint in patients with a frozen shoulder, thus enhancing the treatment effect. To compare the effects of intraarticular injection of lidocaine plus physiotherapy to that of physiotherapy alone in the treatment of a frozen shoulder, a prospective randomized controlled trial was conducted in the rehabilitation department of a private teaching hospital. Patients with a frozen shoulder were randomized into the physiotherapy group or the lidocaine injection plus physiotherapy (INJPT) group. The subjects in the INJPT group underwent injection of 3 ml of 1% lidocaine into the affected shoulder 10 to 20 minutes before each physiotherapy session. In each group, the treatment lasted 3 months. The primary outcome measures were the active and passive range of motion of the affected shoulder. The secondary outcome measures were the results of the Shoulder Disability Questionnaire, the Shoulder Pain and Disability Index, and the 36-item Short-Form Health Survey (SF-36). The outcome measures were evaluated before treatment and 1, 2, 3, 4, and 6 months after the start of treatment. The group comparisons showed significantly greater improvement in the INJPT group, mainly in active and passive shoulder range of motion in flexion and external rotation and improvements in pain and disability (P < 0.05); however, no significant group difference was seen in the SF-36 results. The intraarticular injection of lidocaine immediately before a physiotherapy session might be superior to physiotherapy alone in the treatment of a frozen shoulder.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01817348","term_id":"NCT01817348"}}NCT01817348     

The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.     

Initiating Antiretroviral Therapy for HIV at a Patient’s First Clinic Visit: The RapIT Randomized Controlled Trial

BackgroundHigh rates of patient attrition from care between HIV testing and antiretroviral therapy (ART) initiation have been documented in sub-Saharan Africa, contributing to persistently low CD4 cell counts at treatment initiation. One reason for this is that starting ART in many countries is a lengthy and burdensome process, imposing long waits and multiple clinic visits on patients. We estimated the effect on uptake of ART and viral suppression of an accelerated initiation algorithm that allowed treatment-eligible patients to be dispensed their first supply of antiretroviral medications on the day of their first HIV-related clinic visit.ConclusionsOffering single-visit ART initiation to adult patients in South Africa increased uptake of ART by 36% and viral suppression by 26%. This intervention should be considered for adoption in the public sector in Africa.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01710397","term_id":"NCT01710397"}}NCT01710397, and South African National Clinical Trials Register DOH-27-0213-4177.     

Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and Findings

Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073).     

Genome sequence and description of Aeromicrobium massiliense sp. nov.

Aeromicrobium massiliense strain JC14^Tsp. nov. is the type strain of Aeromicrobium massiliense sp. nov., a new species within the genus Aeromicrobium. This strain, whose genome is described here, was isolated from the fecal microbiota of an asymptomatic patient. Aeromicrobium massiliense is an aerobic rod-shaped gram-positive bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,322,119 bp long genome contains 3,296 protein-coding and 51 RNA genes.     

Scheduled Intermittent Screening with Rapid Diagnostic Tests and Treatment with Dihydroartemisinin-Piperaquine versus Intermittent Preventive Therapy with Sulfadoxine-Pyrimethamine for Malaria in Pregnancy in Malawi: An Open-Label Randomized Controlled Trial

BackgroundIn Africa, most plasmodium infections during pregnancy remain asymptomatic, yet are associated with maternal anemia and low birthweight. WHO recommends intermittent preventive therapy in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). However, sulfadoxine-pyrimethamine (SP) efficacy is threatened by high-level parasite resistance. We conducted a trial to evaluate the efficacy and safety of scheduled intermittent screening with malaria rapid diagnostic tests (RDTs) and treatment of RDT-positive women with dihydroartemisinin-piperaquine (DP) as an alternative strategy to IPTp-SP.ConclusionsScheduled screening for malaria parasites with the current generation of RDTs three to four times during pregnancy as part of focused antenatal care was not superior to IPTp-SP in this area with high malaria transmission and high SP resistance and was associated with higher fetal loss and more malaria at delivery.

Trial Registration

Pan African Clinical Trials Registry PACTR201103000280319; ISRCTN Registry ISRCTN69800930     

Functional group and substructure searching as a tool in metabolomics

Background

A direct link between the names and structures of compounds and the functional groups contained within them is important, not only because biochemists frequently rely on literature that uses a free-text format to describe functional groups, but also because metabolic models depend upon the connections between enzymes and substrates being known and appropriately stored in databases.

Methodology

We have developed a database named “Biochemical Substructure Search Catalogue” (BiSSCat), which contains 489 functional groups, >200,000 compounds and >1,000,000 different computationally constructed substructures, to allow identification of chemical compounds of biological interest.

Conclusions

This database and its associated web-based search program (http://bisscat.org/) can be used to find compounds containing selected combinations of substructures and functional groups. It can be used to determine possible additional substrates for known enzymes and for putative enzymes found in genome projects. Its applications to enzyme inhibitor design are also discussed.