Immunochemical detection of DNA in alkaline sucrose gradient fractions

A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.     

Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

Background: Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the ?13910C?>?T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants.

Methods: We genotyped 3395 routine samples using real-time PCR for the ?13910C?>?T-variant. All consecutive samples identified as ?13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis.

Results: Using real-time PCR resulted in 100% successful genotyping of the ?13910C?>?T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than ?13910C?>?T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%.

Conclusions: We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.     

Awareness of Medicare Regulation Changes: Occupational Therapists' Perceptions and Implications for Practice

Aims: Occupational therapists who work with Medicare beneficiaries should keep abreast about Medicare regulations relevant to practice. This study identified how occupational therapists employed by contractual companies working in skilled nursing facilities (SNFs) learn and interpret changes about Medicare regulations impacting practice. Methods: Researchers analyzed qualitative data from 11 occupational therapists employed in a Midwest metropolitan area through an open-ended questionnaire, focus group, and phone interview. Results: Major themes identified included primary reliance on company resources to learn about Medicare regulation changes, reasons therapists were for or against utilization of noncompany resources for enhanced awareness, barriers to, and suggestions for deepening understanding of Medicare regulation changes. Conclusions: Findings have important implications for the occupational therapy field by contributing to better understanding of how current communication and interpretation regarding Medicare regulations occurs with therapists employed in contracted SNFs and conceivable ways for occupational therapists to gain deeper understanding of Medicare regulations impacting practice.     

Defective T suppressor-inducer cell function in human immune deficiency virus-seropositive hemophilia patients

In human immune deficiency virus (HIV)-seropositive hemophilia patients, a low number of CD4 + lymphocytes is found, as well as a low CD4+/CD8+ ratio. In previous studies, it has been shown that antigen- specific T-helper cell (CD4+) function was present and no excessive antigen-specific T-suppressor cell (CD8+) function could be demonstrated. In this report, we studied another activity of CD4+ cells, namely the capacity to induce T-suppressor cell activity. The results clearly show a selective dysfunction of CD4+ suppressor-inducer (Tsi) cell function. Since these HIV-seropositive hemophilia patients showed the presence of activated B cells in the peripheral circulation refractory to antigen-specific T-helper cell signals and secreting specific antibodies spontaneously, we raised the hypothesis that the activated B cells in the patients activate the Tsi cells in vivo. This constant activation leads to a functional exhaustion of the Tsi cell pool.     

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss andduplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival andmay represent an initiating event in tumorigenesis.     

Recovery of T cell subsets after autologous bone marrow transplantation is mainly due to proliferation of mature T cells in the graft

In 22 patients with malignancies, treated with high-dose chemoradiotherapy and autologous bone marrow transplantation (BMT), peripheral blood T cell subsets and functions were studied. In ten cytomegalovirus (CMV)-negative patients, CD4+ and CD8+ T cells (representing T cells of the helper/inducer phenotype and T cells of the suppressor/cytotoxic phenotype, respectively), recovered slowly and simultaneously. In 12 CMV-positive patients, however, CD8+ T cells recovered more rapidly than CD4+ T cells and rose to increased counts. No T cells with an immature phenotype (CD1+, OKT6+) were observed. Lymphocyte stimulation by herpes simplex virus infected fibroblasts (and by CMV-infected fibroblasts in CMV-positive patients) in contrast remained high and even increased after BMT in both groups. These data indicate that T cell recovery after autologous BMT is mainly due to proliferation of mature T cells present in the BM graft and not to generation of new T cells from T cell precursors.     

Lower levels of maternal capital in early life predict offspring obesity in adulthood

Background: As of 2013, 65% of the world’s population lived in countries where overweight/obesity kills more people than being underweight. Evolutionary perspectives provide a holistic understanding of both how and why obesity develops and its long-term implications.

Aim: To test whether the maternal capital hypothesis, an evolutionary perspective, is viable for explaining the development of obesity in adulthood.

Subjects and methods: Restricted-use data from the National Longitudinal Study of Adolescent Health (Add Health; n?=?11?403) was analysed using logistic regressions. The sample included adolescents and their biological mothers.

Results: The odds of obesity in adulthood increased by 22% for every standard deviation increase in lack of maternal capital (Exp (B)?=?1.22, p?Conclusion: The maternal capital perspective is useful for explaining how and why early life characteristics (including maternal resources) predict obesity in adulthood. Implications of the findings are discussed.     

Evaluation of Portability and Cost of a Fluorescent PCR Ribotyping Protocol for Clostridium difficile Epidemiology

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.     

CC and CXC chemokines in breastmilk are associated with mother-to-child HIV-1 transmission

INTRODUCTION: CC and CXC chemokines may play a role in mother-to-child HIV-1 transmission by blocking HIV-1 binding to chemokine receptors and impeding viral entry into cells. METHODS: To define correlates of breastmilk chemokines and associations with infant HIV-1 acquisition, chemokines in breastmilk and infant HIV-1 infection risk were assessed in an observational, longitudinal cohort study. We measured MIP-1alpha, MIP-1beta, RANTES, and SDF-1 in month 1 breastmilk specimens from HIV-1-infected women in Nairobi and HIV-1 viral load was calculated in maternal plasma and breastmilk at delivery and 1 month postpartum. Infant infection status was determined at birth and months 1, 3, 6, 9, and 12. RESULTS: Among 281 breastfeeding women, 60 (21%) of their infants acquired HIV-1 during follow-up, 39 (65%) of whom became infected intrapartum or after birth. MIP-1alpha, MIP-1beta, RANTES, and SDF-1 were all positively correlated with breastmilk HIV-1 RNA (P<0.0005). Women with clinical mastitis had 50% higher MIP-1alpha and MIP-1beta levels (P<0.001 and P=0.006, respectively) and women with subclinical mastitis (breastmilk Na(+)/K(+)>1) had approximately 70% higher MIP-1alpha, MIP-1beta and RANTES (P<0.002 for all) compared to women without mastitis. Independent of breastmilk HIV-1, increased MIP-1beta and SDF-1 were associated with reduced risk of infant HIV-1 (RR=0.4; 95% CI 0.2-0.9; P=0.03 and RR=0.5; 95% CI=0.3-0.9; P=0.02, respectively) and increased RANTES was associated with higher transmission risk (RR=2.3; 95% CI 1.1- 5.3; P=0.04). CONCLUSIONS: These observations suggest a complex interplay between virus levels, breastmilk chemokines, and mother-to-child HIV-1 transmission and may provide insight into developing novel strategies to reduce infection across mucosal surfaces.