Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS

BACKGROUND: The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures. RESULTS: We have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1 × 10(7 )cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weight differences based on existing protein annotation. CONCLUSION: We demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain

Loss-of-function mutations of NaV1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of NaV1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of NaV1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of NaV1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with NaV1.7 inhibitors and significantly reduced in NaV1.7−/− mice. To validate the use of the model for profiling NaV1.7 inhibitors, we determined the NaV selectivity and tested the efficacy of the reported NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited NaV1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited NaV channels and was only effective in the OD1 model when delivered systemically. Our novel model of NaV1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of NaV1.7 inhibitors

Effects of 3-Weeks of High-Intensity Interval Training on Running Economy and Endurance

Please view abstract in the attached PDF fil

Prenatal DDT Exposure in Relation to Anthropometric and Pubertal Measures in Adolescent Males

DDT (dichlorodiphenyltrichloroethane), a pesticide once used widely in agriculture and now limited to public health use, remains a controversial chemical because of a combination of benefits and risks. DDT or its breakdown products are ubiquitous in the environment and in humans. Compounds in the DDT family have endocrine actions and have been associated with reproductive toxicity. A previous study reported associations between prenatal exposure to p,p′-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene] and increased height and weight in adolescent boys. We examined a group with higher exposures to see whether similar associations would occur. Our study group was 304 males born in Philadelphia in the early 1960s who had participated in a previous study. Anthropometric and pubertal measures from one to six visits during their adolescent years were available, as were stored maternal serum samples from pregnancy. We measured p,p′-DDE, p,p′-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane], and o,p′-DDT [1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)-ethane] in the maternal serum. Outcomes examined in the boys were height, ratio of sitting height to height, body mass index, triceps skinfold thickness, ratio of subscapular to the sum of triceps and subscapular skinfold thicknesses, skeletal age, serum testosterone, and serum dehydroepiandrosterone sulfate. No associations between prenatal exposure to any of the DDT compounds and any outcome measure were seen

Maternal risk factors for abnormal placental growth: The national collaborative perinatal project

<p>Abstract</p> <p>Background</p> <p>Previous studies of maternal risk factors for abnormal placental growth have focused on placental weight and placental ratio as measures of placental growth. We sought to identify maternal risk factors for placental weight and two neglected dimensions of placental growth: placental thickness and chorionic plate area.</p> <p>Methods</p> <p>We conducted an analysis of 24,135 mother-placenta pairs enrolled in the National Collaborative Perinatal Project, a prospective cohort study of pregnancy and child health. We defined growth restriction as < 10^thpercentile and hypertrophy as > 90^thpercentile for three placental growth dimensions: placental weight, placental thickness and chorionic plate area. We constructed parallel multinomial logistic regression analyses to identify (a) predictors of restricted growth (vs. normal) and (b) predictors of hypertrophic growth (vs. normal).</p> <p>Results</p> <p>Black race was associated with an increased likelihood of growth restriction for placental weight, thickness and chorionic plate area, but was associated with a reduced likelihood of hypertrophy for these three placental growth dimensions. We observed an increased likelihood of growth restriction for placental weight and chorionic plate area among mothers with hypertensive disease at 24 weeks or beyond. Anemia was associated with a reduced likelihood of growth restriction for placental weight and chorionic plate area. Pre-pregnancy BMI and pregnancy weight gain were associated with a reduced likelihood of growth restriction and an increased likelihood of hypertrophy for all three dimensions of placental growth.</p> <p>Conclusion</p> <p>Maternal risk factors are either associated with placental growth restriction or placental hypertrophy not both. Our findings suggest that the placenta may have compensatory responses to certain maternal risk factors suggesting different underlying biological mechanisms.</p

Nicotine Acts on Growth Plate Chondrocytes to Delay Skeletal Growth through the α7 Neuronal Nicotinic Acetylcholine Receptor

BACKGROUND: Cigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking-induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA), a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/- mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR -/- fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR. CONCLUSIONS/SIGNIFICANCE: These results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease matrix synthesis, suppress hypertrophic differentiation via alpha7 nAChR, leading to delayed skeletal growth

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution

<p>Abstract</p> <p>Background</p> <p>Group B <it>Streptococcus </it>(GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations.</p> <p>Methods</p> <p>To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence.</p> <p>Results</p> <p>Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005).</p> <p>Conclusion</p> <p>This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.</p

Head start teaching center: Outcome evaluation of 3 years of participatory training

The design and evaluation of the New England Head Start Teaching Center (NEHSTC), one of 14 federally funded programs created to test the efficacy of participatory, hands-on training for enhancing Head Start service delivery is the focus of this article. The unique characteristics of the NEHSTC and the outcome evaluation results from 3 years of training will be discussed. The findings demonstrate the NEHSTC was successful in implementing high quality, participatory training within the context of an ongoing Head Start program. Various Head Start staff who participated in the NEHSTC trainings demonstrated gains in knowledge, skills, and attitudes compared to similar Head Start employees who did not receive training. The positive findings suggest that participatory training should be included in the menu of training options available. Because of the unique size and scope of Head Start, the success of ongoing efforts to improve the quality of its programs and services are particularly significant. Within Head Start, this discussion of quality enhancements via innovative training models is timely given the advent of the new performance standards and the restructuring of the Training and Technical Assistance system. Additionally, the findings are relevant for broader efforts to improve early care and education programs nationwide. © 1998 Taylor & Francis Group, LLC

Head start teaching center: Evaluation of a new approach to head start staff development

Head Start Teaching Centers are a national demonstration project designed to provide participatory training in all Head Start component areas within the context of an exemplary Head Start program. Each Teaching Center employs an independent evaluation to study this alternative approach to Head Start staff development. This paper presents the results of the outcome evaluation for the first year of the New England Head Start Teaching Center. The New England Head Start Teaching Center was designed to provide intensive training during a 3 or 5 day period of residence at the Teaching Center. This paper briefly describes the national Head Start Teaching Center model, the implementation of this model in the New England region, the outcome evaluation plan, and the results from the first year of training. The analyses of year one data indicated that training provided by the New England Head Start Teaching Center produced significant gains. As compared to similar Head Start employees who did not participate in training, both trainees and their supervisors reported significant gains in trainees\u27 knowledge, skills, and expertise after participating in the New England Head Start Teaching Center training. © 1997 Ablex Publishing Corp. All rights of reproduction reserved