Efficient, high-throughput transfection of human embryonic stem cells

INTRODUCTION: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. METHODS: We investigated the ability of two commercially available electroporation systems, the Nucleofection(® )96-well Shuttle(® )System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. RESULTS: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection(® )96-well Shuttle(® )System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. CONCLUSIONS: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology

Eff ectiveness of the 13-valent pneumococcal conjugate vaccine against invasive pneumococcal disease in South African children: a case-control study

Background The 13-valent pneumococcal conjugate vaccine (PCV13) was designed to include disease-causing serotypes that are important in low-income and middle-income countries. Vaccine eff ectiveness estimates are scarce in these settings. South Africa replaced PCV7 with PCV13 in 2011 using a 2 + 1 schedule. We aimed to assess the eff ectiveness of two or more doses of PCV13 against invasive pneumococcal disease in children with HIV infection and in those not infected with HIV. Methods Cases of invasive pneumococcal disease in children aged 5 years or younger were identifi ed through national laboratory-based surveillance. Isolates were serotyped with the Quellung reaction or PCR. We sought in-hospital controls for every case, matched for age, HIV status, and study site. We aimed to enrol four controls for every case not infected with HIV and six controls for every case with HIV infection (case-control sets). With conditional logistic regression, we calculated vaccine eff ectiveness as a percentage, with the equation 1 – [adjusted odds ratio for vaccination] × 100. We included data from an earlier investigation of PCV7 to assess vaccine eff ectiveness in children exposed to but not infected with HIV and in malnourished children not infected with HIV. Findings Between January, 2012, and December, 2014, we enrolled children aged 16 weeks or older to our study: 240 were cases not infected with HIV, 75 were cases with HIV infection, 1118 were controls not infected with HIV, and 283 were controls with HIV infection. The eff ectiveness of two or more doses of PCV13 against PCV13-serotype invasive pneumococcal disease was 85% (95% CI 37 to 96) among 11 case-control sets of children not infected with HIV and 91% (–35 to 100) among three case-control sets of children with HIV infection. PCV13 eff ectiveness among 26 case-control sets of children not infected with HIV was 52% (95% CI –12 to 79) against all-serotype invasive pneumococcal disease and 94% (44 to 100) for serotype 19A. Vaccine eff ectiveness against PCV7-serotype invasive pneumococcal disease was 87% (95% CI 38 to 97) in children exposed to HIV but uninfected and 90% (53 to 98) in malnourished children not infected with HIV. Interpretation Our results indicate that PCV13 in a 2 + 1 schedule is eff ective for preventing vaccine-type pneumococcal infections in young children not infected with HIV, including those who are malnourished or who have been exposed to HIV. Although the point estimate for PCV13 vaccine eff ectiveness in children infected with HIV was high, it did not reach signifi cance, possibly because of the small sample size. These fi ndings support recommendations for widespread use of pneumococcal conjugate vaccine in low-income and middle-income countries

Screening Yield of HIV Antigen/Antibody Combination and Pooled HIV RNA Testing for Acute HIV Infection in a High-Prevalence Population

Although acute HIV infection contributes disproportionately to onward HIV transmission, HIV testing has not routinely included screening for acute HIV infection. To evaluate the performance of an HIV antigen/antibody (Ag/Ab) combination assay to detect acute HIV infection compared with pooled HIV RNA testing. Multisite, prospective, within-individual comparison study conducted between September 2011 and October 2013 in 7 sexually transmitted infection clinics and 5 community-based programs in New York, California, and North Carolina. Participants were 12 years or older and seeking HIV testing, without known HIV infection. All participants with a negative rapid HIV test result were screened for acute HIV infection with an HIV Ag/Ab combination assay (index test) and pooled human immunodeficiency virus 1 (HIV-1) RNA testing. HIV RNA testing was the reference standard, with positive reference standard result defined as detectable HIV-1 RNA on an individual RNA test. Number and proportion with acute HIV infections detected. Among 86,836 participants with complete test results (median age, 29 years; 75.0% men; 51.8% men who have sex with men), established HIV infection was diagnosed in 1158 participants (1.33%) and acute HIV infection was diagnosed in 168 participants (0.19%). Acute HIV infection was detected in 134 participants with HIV Ag/Ab combination testing (0.15% [95% CI, 0.13%-0.18%]; sensitivity, 79.8% [95% CI, 72.9%-85.6%]; specificity, 99.9% [95% CI, 99.9%-99.9%]; positive predictive value, 59.0% [95% CI, 52.3%-65.5%]) and in 164 participants with pooled HIV RNA testing (0.19% [95% CI, 0.16%-0.22%]; sensitivity, 97.6% [95% CI, 94.0%-99.4%]; specificity, 100% [95% CI, 100%-100%]; positive predictive value, 96.5% [95% CI, 92.5%-98.7%]; sensitivity comparison, P < .001). Overall HIV Ag/Ab combination testing detected 82% of acute HIV infections detectable by pooled HIV RNA testing. Compared with rapid HIV testing alone, HIV Ag/Ab combination testing increased the relative HIV diagnostic yield (both established and acute HIV infections) by 10.4% (95% CI, 8.8%-12.2%) and pooled HIV RNA testing increased the relative HIV diagnostic yield by 12.4% (95% CI, 10.7%-14.3%). In a high-prevalence population, HIV screening using an HIV Ag/Ab combination assay following a negative rapid test detected 82% of acute HIV infections detectable by pooled HIV RNA testing, with a positive predictive value of 59%. Further research is needed to evaluate this strategy in lower-prevalence populations and in persons using preexposure prophylaxis for HIV prevention

Health- and oral health-related quality of life among preschool children with cerebral palsy

Objectives: To assess the health- and oral health-related quality of life of preschool children with cerebral palsy (CP) and to determine their inter-relationship between the two quality of life measures. Methods: A total of 144 preschool children with and without CP were invited to participate in the case-control study. Health-related quality of life was assessed by the Pediatric Quality of Life Inventory Version 4.0 (PedsQL™ 4.0) and oral health-related quality of life by the Early Childhood Oral Health Impact Scale (ECOHIS). Differences in PedsQL™ 4.0 and ECOHIS scores were determined between the groups, and correlation between PedsQL and ECOHIS were explored. Results: Significant differences in overall scores of PedsQL™ 4.0 (P < 0.001) and in overall scores of ECOHIS (P < 0.05) were apparent between the two groups. In terms of health- and oral health-related quality of life, preschool children with CP fared worse than the age-gender-matched control group. There was a positive albeit weak correlation (r = 0.203, P < 0.05) between PedsQL™ 4.0 and ECOHIS scores. Conclusions: Differences in health- and oral health-related quality of life exist among preschool children with CP. Correlation between health- and oral health-related quality of life could at best be described as weak. © 2010 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Estimated severe pneumococcal disease cases and deaths before and after pneumococcal conjugate vaccine introduction in children younger than 5 years of age in South Africa

INTRODUCTION : Streptococcus pneumoniae is a leading cause of severe bacterial infections globally. A full understanding of the impact of pneumococcal conjugate vaccine (PCV) on pneumococcal disease burden, following its introduction in 2009 in South Africa, can support national policy on PCV use and assist with policy decisions elsewhere. METHODS : We developed a model to estimate the national burden of severe pneumococcal disease, i.e. disease requiring hospitalisation, pre- (2005±2008) and post-PCV introduction (2012± 2013) in children aged 0±59 months in South Africa. We estimated case numbers for invasive pneumococcal disease using data from the national laboratory-based surveillance, adjusted for specimen-taking practices. We estimated non-bacteraemic pneumococcal pneumonia case numbers using vaccine probe study data. To estimate pneumococcal deaths, we applied observed case fatality ratios to estimated case numbers. Estimates were stratified by HIV status to account for the impact of PCV and HIV-related interventions. We assessed how different assumptions affected estimates using a sensitivity analysis. Bootstrapping created confidence intervals. RESULTS : In the pre-vaccine era, a total of approximately 107,600 (95% confidence interval [CI] 83,000±140,000) cases of severe hospitalised pneumococcal disease were estimated to have occurred annually. Following PCV introduction and the improvement in HIV interventions, 41,800 (95% CI 28,000±50,000) severe pneumococcal disease cases were estimated in 2012±2013, a rate reduction of 1,277 cases per 100,000 child-years. Approximately 5000 (95% CI 3000±6000) pneumococcal-related annual deaths were estimated in the prevaccine period and 1,900 (95% CI 1000±2500) in 2012±2013, a mortality rate difference of 61 per 100,000 child-years. CONCLUSIONS : While a large number of hospitalisations and deaths due to pneumococcal disease still occur among children 0±59 months in South Africa, we found a large reduction in this estimate that is temporally associated with PCV introduction. In HIV-infected individuals the scale-up of other interventions, such as improvements in HIV care, may have also contributed to the declines in pneumococcal burden.S1 Text. Supplementary material: Estimated severe pneumococcal disease cases and deaths before and after pneumococcal conjugate vaccine introduction in children younger than 5 years of age in South Africa.S1 Table. Population denominators from the Thembisa model for children <5 years of age in South Africa, 2005-2008 and 2012-2013.S2 Table. Sensitivity analysis for case numbers showing key variables altered in analysis, 2005-2008 and 2012-2013.S3 Table. Sensitivity analysis for numbers of deaths showing key variables altered in analysis, 2005-2008 and 2012-2013.S1 Fig. Initial step in estimating the burden of invasive and non-invasive pneumococcal cases in children aged <5 years in South Africa, 2005-2008 and 2012-2013.S2 Fig. Second step in estimating the burden of invasive and non-invasive pneumococcal cases in children <5 years in South Africa, 2005-2008 and 2012-2013.S3 Fig. Tornado sensitivity diagram representing change in pneumococcal case estimates in children <5 years of age in the pre-vaccine era, when values of key variables are modified.S4 Fig. Tornado sensitivity diagram representing change in pneumococcal death estimates in children <5 years of age in the pre-vaccine era, when values of key variables are modified.The National Institute for Communicable Diseases/National Health Laboratory Service (NICD/NHLS), South Africa and the Centers for Disease Control and Prevention (CDC) Global AIDS Program (GAP) Cooperative Agreement (U62/PSO022901).http://www.plosone.orgam2017Paediatrics and Child Healt

Psoriasis and Hypertension Severity: Results from a Case-Control Study

BACKGROUND: Epidemiologic studies have provided new insights into the association between psoriasis and cardiovascular diseases. Previous population studies have examined hypertension frequency in psoriasis patients. However, the relationship between severity of hypertension and psoriasis has not been characterized. OBJECTIVE: We sought to investigate whether patients with psoriasis have more difficult-to-manage hypertension compared to non-psoriatic hypertensive patients. APPROACH: We performed a case-control study using the University of California Davis electronic medical records. The cases were defined as patients diagnosed with both psoriasis and hypertension, and controls were defined as patients with hypertension and without psoriasis. In this identified population, 835 cases were matched on age, sex, and body mass index (BMI) to 2418 control patients. KEY RESULTS: Treatment with multiple anti-hypertensives was significantly associated with the presence of psoriasis using univariate (p < 0.0001) and multivariable analysis, after adjusting for diabetes, hyperlipidemia, and race (p < 0.0001). Compared to hypertensive patients without psoriasis, psoriasis patients with hypertension were 5 times more likely to be on a monotherapy antihypertensive regimen (95% CI 3.607.05), 9.5 times more likely to be on dual antihypertensive therapy (95% CI 6.68-13.65), 16.5 times more likely to be on triple antihypertensive regimen (95% CI 11.01-24.84), and 19.9 times more likely to be on quadruple therapy or centrally-acting agent (95% CI 10.58-37.33) in multivariable analysis after adjusting for traditional cardiac risk factors. CONCLUSIONS: Psoriasis patients appear to have more difficult-to-control hypertension compared to non-psoriatic, hypertensive patients

Efficacy of Structural-Level Condom Distribution Interventions: A Meta-Analysis of U.S. and International Studies, 1998–2007

This systematic review examines the overall efficacy of U.S. and international-based structural-level condom distribution interventions (SLCDIs) on HIV risk behaviors and STIs and identifies factors associated with intervention efficacy. A comprehensive literature search of studies published from January 1988 through September 2007 yielded 21 relevant studies. Significant intervention effects were found for the following outcomes: condom use, condom acquisition/condom carrying, delayed sexual initiation among youth, and reduced incident STIs. The stratified analyses for condom use indicated that interventions were efficacious for various groups (e.g., youth, adults, males, commercial sex workers, clinic populations, and populations in areas with high STI incidence). Interventions increasing the availability of or accessibility to condoms or including additional individual, small-group or community-level components along with condom distribution were shown to be efficacious in increasing condom use behaviors. This review suggests that SLCDIs provide an efficacious means of HIV/STI prevention

Measures of Association for Identifying MicroRNA-mRNA Pairs of Biological Interest

MicroRNAs are a class of small non-protein coding RNAs that play an important role in the regulation of gene expression. Most studies on the identification of microRNA-mRNA pairs utilize the correlation coefficient as a measure of association. The use of correlation coefficient is appropriate if the expression data are available for several conditions and, for a given condition, both microRNA and mRNA expression profiles are obtained from the same set of individuals. However, there are many instances where one of the requirements is not satisfied. Therefore, there is a need for new measures of association to identify the microRNA-mRNA pairs of interest and we present two such measures. The first measure requires expression data for multiple conditions but, for a given condition, the microRNA and mRNA expression may be obtained from different individuals. The new measure, unlike the correlation coefficient, is suitable for analyzing large data sets which are obtained by combining several independent studies on microRNAs and mRNAs. Our second measure is able to handle expression data that correspond to just two conditions but, for a given condition, the microRNA and mRNA expression must be obtained from the same set of individuals. This measure, unlike the correlation coefficient, is appropriate for analyzing data sets with a small number of conditions. We apply our new measures of association to multiple myeloma data sets, which cannot be analyzed using the correlation coefficient, and identify several microRNA-mRNA pairs involved in apoptosis and cell proliferation

Risk Factors for Hospitalizations Among Older Adults with Gastrointestinal Cancers

Background: Older adults (≥65 years) with gastrointestinal (GI) cancers who receive chemotherapy are at increased risk of hospitalization caused by treatment-related toxicity. Geriatric assessment (GA) has been previously shown to predict risk of toxicity in older adults undergoing chemotherapy. However, studies incorporating the GA specifically in older adults with GI cancers have been limited. This study sought to identify GA-based risk factors for chemotherapy toxicity-related hospitalization among older adults with GI cancers. Patients and methods: We performed a secondary post hoc subgroup analysis of two prospective studies used to develop and validate a GA-based chemotherapy toxicity score. The incidence of unplanned hospitalizations during the course of chemotherapy treatment was determined. Results: This analysis included 199 patients aged ≥65 years with a diagnosis of GI cancer (85 colorectal, 51 gastric/esophageal, and 63 pancreatic/hepatobiliary). Sixty-five (32.7%) patients had ≥1 hospitalization. Univariate analysis identified sex (female), cardiac comorbidity, stage IV disease, low serum albumin, cancer type (gastric/esophageal), hearing deficits, and polypharmacy as risk factors for hospitalization. Multivariable analyses found that patients who had cardiac comorbidity (OR 2.48, 95% CI 1.13-5.42) were significantly more likely to be hospitalized. Conclusion: Cardiac comorbidity may be a risk factor for hospitalization in older adults with GI cancers receiving chemotherapy. Further studies with larger sample sizes are warranted to examine the relationship between GA measures and hospitalization in this vulnerable population