BACKGROUND: Telephone counseling Quitlines can support smoking cessation, but are under-utilized. We explored the use of smoker peer-referrals to increase use of a Quitline in Mississippi and Alabama.
FINDINGS: Collaborating with the Alabama and Mississippi Quitline, we piloted peer-referrals to Quitlines. Successful 'quitters' who had used the Quitline were contacted at routine follow-up and recruited to participate as a peer-referrer and refer their friends and family who smoked to the Quitline. Peer-referrers completed a training session, received a manual and a set of Quitline brochures a peer-referral forms. These peer-referral forms were then returned to the Quitline telephone counselors who proactively called the referred smokers. Of the initial potential pool of 96 who quit using the Quitline, 24 peer-referrers (75% Women, 29% African-American, and high school graduates/GED 67%) were recruited and initially agreed to participate as peer-referrers. Eleven of the 24 who initially agreed were trained, and of these 11, 4 (4%) actively referred 23 friends and family over 2 months. From these 23 new referrals, three intakes (100% Women, 66% African-American) were completed. Of the initial pool of 96, 4 (4%) actively participated in referring friends and family. Quitline staff and peer-referrers noted several barriers including: time-point in which potential peer-referrers were asked to participate, an 'overwhelming' referral form to use and limited ways to refer.
CONCLUSIONS: Though 'quitters' were willing to agree to peer-refer, we received a minority of referrals. However, we identified several areas to improve this new method for increasing awareness and access to support systems like the Quitline for smokers who want to quit.
Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
BACKGROUND: The human peptidyl-prolyl isomerase Cyclophilin A (CypA) binds HIV-1 capsid (CA) and influences early steps in the HIV-1 replication cycle. The mechanism by which CypA regulates HIV-1 transduction efficiency is unknown. Disruption of CypA binding to CA, either by genetic means or by the competitive inhibitor cyclosporine A (CsA), reduces the efficiency of HIV-1 transduction in some cells but not in others. Transduction of certain cell types increases significantly when CypA binding to particular HIV-1 CA mutants, i.e., A92E, is prevented. Previous studies have suggested that this cell type-specific effect is due to a dominant-acting, CypA-dependent restriction factor.
RESULTS: Here we investigated the mechanism by which CypA regulates HIV-1 transduction efficiency using 27 different human cell lines, 32 HeLa subclones, and several previously characterized HIV-1 CA mutants. Disruption of CypA binding to wild-type CA, or to any of the mutant CAs, caused a decrease in HIV-1 reverse transcription in all the cell lines analyzed here. This block to reverse transcription, though, did not correlate with cell type-specific effects on transduction efficiency. The level of 2-LTR circles, a marker for nuclear transport of the viral cDNA that results from reverse transcription, correlated closely with effects on infectivity. No correlation was observed between the cell type-specific effects on infectivity and the steady-state CypA protein levels in these cells. Instead, as indicated by a fate-of-capsid assay, CsA released the HIV-1 CA core from an apparent state of hyperstabilization, in a cell type-specific manner.
CONCLUSION: These data demonstrate that, while CypA promotes reverse transcription under all conditions tested here, its effect on HIV-1 infectivity correlates more closely with effects on nuclear entry of the viral cDNA. The data also support the hypothesis that a cell-type specific CypA-dependent restriction factor blocks HIV-1 replication by delaying CA core uncoating and hindering nuclear entry.
A novel rabbit monoclonal antibody platform to dissect the diverse repertoire of antibody epitopes for HIV-1 Env immunogen design
The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines.
Estudio Parto: postpartum diabetes prevention program for hispanic women with abnormal glucose tolerance in pregnancy: a randomised controlled trial - study protocol
BACKGROUND: Diabetes and obesity have reached epidemic proportions in the U.S. with rates consistently higher among Hispanics as compared to non-Hispanic whites. Among Hispanic women diagnosed with gestational diabetes mellitus (GDM), 50% will go on to develop type 2 diabetes within 5 years of the index pregnancy. Although randomised controlled trials among adults with impaired glucose tolerance have shown that diet and physical activity reduce the risk of type 2 diabetes, such programs have not been tested in high-risk postpartum women. The overall goal of this randomised controlled trial is to test the efficacy of a culturally and linguistically modified, individually-tailored lifestyle intervention to reduce risk factors for type 2 diabetes and cardiovascular disease among postpartum Hispanic women with a history of abnormal glucose tolerance during pregnancy.
METHODS/DESIGN: Hispanic pregnant women who screen positive for GDM will be recruited and randomly assigned to a Lifestyle Intervention (n = 150) or a Health and Wellness (control) Intervention (n = 150). Multimodal contacts (i.e., in-person, telephone, and mailed materials) will be used to deliver the intervention from late pregnancy (29 weeks gestation) to 12 months postpartum. Targets of the intervention are to achieve Institute of Medicine Guidelines for postpartum weight loss; American Congress of Obstetrician and Gynecologist guidelines for physical activity; and American Diabetes Association guidelines for diet. The intervention draws from Social Cognitive Theory and the Transtheoretical Model and addresses the specific cultural and environmental challenges faced by low-income Hispanic women. Assessments will be conducted at enrollment, and at 6-weeks, 6-months, and 12-months postpartum by trained bicultural and bilingual personnel blinded to the intervention arm. Efficacy will be assessed via postpartum weight loss and biomarkers of insulin resistance and cardiovascular risk. Changes in physical activity and diet will be measured via 7-day actigraph data and three unannounced 24-hour dietary recalls at each assessment time period.
DISCUSSION: Hispanic women are the fastest growing minority group in the U.S. and have the highest rates of sedentary behavior and postpartum diabetes after a diagnosis of GDM. This randomised trial uses a high-reach, low-cost strategy that can readily be translated into clinical practice in underserved and minority populations.
TRIAL REGISTRATION: NCT01679210.
Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA
BACKGROUND: All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated.
RESULTS: The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis.
CONCLUSION: These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.
BACKGROUND: In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive.
RESULTS: Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 A resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity.
CONCLUSIONS: While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.