Validating Candidate Congenital Heart Disease Genes in Drosophila.

Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate congenital heart disease (CHD) genes. The protocols exploit comprehensive libraries of UAS-GeneX-RNAi fly strains that when crossed into a 4×Hand-Gal4 genetic background afford highly efficient cardiac-specific knockdown of endogenous fly orthologs of human genes. A panel of quantitative assays evaluates phenotypic severity across multiple cardiac parameters. These include developmental lethality, larva and adult heart morphology, and adult longevity. These protocols were recently used to evaluate more than 100 candidate CHD genes implicated by patient whole-exome sequencing (Zhu et al., 2017)

Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum.

BackgroundA vaccine that interrupts malaria transmission (VIMT) would be a valuable tool for malaria control and elimination. One VIMT approach is to identify sexual erythrocytic and mosquito stage antigens of the malaria parasite that induce immune responses targeted at disrupting parasite development in the mosquito. The standard Plasmodium falciparum membrane-feeding assay (SMFA) is used to assess transmission-blocking activity (TBA) of antibodies against candidate immunogens and of drugs targeting the mosquito stages. To develop its P. falciparum sporozoite (SPZ) products, Sanaria has industrialized the production of P. falciparum-infected Anopheles stephensi mosquitoes, incorporating quantitative analyses of oocyst and P. falciparum SPZ infections as part of the manufacturing process.MethodsThese capabilities were exploited to develop a robust, reliable, consistent SMFA that was used to assess 188 serum samples from animals immunized with the candidate vaccine immunogen, Pfs25, targeting P. falciparum mosquito stages. Seventy-four independent SMFAs were performed. Infection intensity (number of oocysts/mosquito) and infection prevalence (percentage of mosquitoes infected with oocysts) were compared between mosquitoes fed cultured gametocytes plus normal human O(+) serum (negative control), anti-Pfs25 polyclonal antisera (MRA39 or MRA38, at a final dilution in the blood meal of 1:54 as positive control), and test sera from animals immunized with Pfs25 (at a final dilution in the blood meal of 1:9).ResultsSMFA negative controls consistently yielded high infection intensity (mean = 46.1 oocysts/midgut, range of positives 3.7-135.6) and infection prevalence (mean = 94.2%, range 71.4-100.0) and in positive controls, infection intensity was reduced by 81.6% (anti-Pfs25 MRA39) and 97.0% (anti-Pfs25 MRA38), and infection prevalence was reduced by 12.9 and 63.5%, respectively. A range of TBAs was detected among the 188 test samples assayed in duplicate. Consistent administration of infectious gametocytes to mosquitoes within and between assays was achieved, and the TBA of anti-Pfs25 control antibodies was highly reproducible.ConclusionsThese results demonstrate a robust capacity to perform the SMFA in a medium-to-high throughput format, suitable for assessing large numbers of experimental samples of candidate antibodies or drugs

Life in Hampton Roads Report: The 12th Annual Life in Hampton Roads Survey

[From the Executive Summary] The Social Science Research Center (SSRC) at Old Dominion University is pleased to present the results from the 12th annual Life in Hampton Roads (LIHR) survey. The purpose of the survey was to gain insight into residents’ perceptions of the quality of life in Hampton Roads. It is important to note that the methodology for this year’s survey differs from previous Life in Hampton Roads surveys. The first ten years of the survey were conducted using a random sample of Hampton Roads residents via telephone. Last year state and university COVID-19 restrictions did not allow for staffing of the SSRC call center during the survey period. Therefore, on-line survey panels were used to solicit respondents to complete a web-based survey. This year, a mixed methods approach of telephone calls and web surveys were used to administer the survey. Given the continued and evolving pandemic conditions in Hampton Roads and the rest of the world, many of this year’s questions focused on residents’ experiences with and responses to continuing COVID-19 conditions

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total HIV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA–positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART

A combination of methods needed to assess the actual use of provisioning ecosystem services

Failure to recognize that potential provisioning ecosystem services are not necessarily collected and used by people may have important consequences for management of land and resources. Accounting for people's actual use of ecosystem services in decision making processes requires a robust methodological approach that goes beyond mapping the presence of ecosystem services. But no such universally accepted method exists, and there are several shortcomings of existing methods such as the application of land use/cover as a proxy for provisioning ecosystem service availability and surveys based on respondents' recall to assess people's collection of e.g. wild food. By combining four complementary methods and applying these to the shifting cultivation systems of Laos, we show how people’s actual use of ecosystem services from agricultural fields differs from ecosystem service availability. Our study is the first in Southeast Asia to combine plot monitoring, collection diaries, repeat interviews, and participant observation. By applying these multiple methods borrowed from anthropology and botany among other research domains, the study illustrates that no single method is sufficient on its own. It is of key importance for scientists to adopt methods that can account for both availability of various services and actual use of those services

Semileptonic B decays to excited charmed mesons

Exclusive semileptonic B decays into excited charmed mesons are investigated at order $\Lambda_{QCD}/m_Q$ in the heavy quark effective theory. Differential decay rates for each helicity state of the four lightest excited $D$ mesons ($D_1$, $D_2^*$, $D_0^*$, and $D_1^*$) are examined. At zero recoil, $\Lambda_{QCD}/m_Q$ corrections to the matrix elements of the weak currents can be written in terms of the leading Isgur-Wise functions for the corresponding transition and meson mass splittings. A model independent prediction is found for the slope parameter of the decay rate into helicity zero $D_1$ at zero recoil. The differential decay rates are predicted, including $\Lambda_{QCD}/m_Q$ corrections with some model dependence away from zero recoil and including order $\alpha_s$ corrections. Ratios of various exclusive branching ratios are computed. Matrix elements of the weak currents between $B$ mesons and other excited charmed mesons are discussed at zero recoil to order $\Lambda_{QCD}/m_Q$. These amplitudes vanish at leading order, and can be written at order $\Lambda_{QCD}/m_Q$ in terms of local matrix elements. Applications to $B$ decay sum rules and factorization are presented.Comment: 39 pages revtex including 10 figures, uses epsf. Substantial improvements throughout the pape

Cerebral oximetry during cardiac arrest : a multicenter study of neurologic outcomes and survival

OBJECTIVES Cardiac arrest is associated with morbidity and mortality because of cerebral ischemia. Therefore, we tested the hypothesis that higher regional cerebral oxygenation during resuscitation is associated with improved return of spontaneous circulation, survival, and neurologic outcomes at hospital discharge. We further examined the validity of regional cerebral oxygenation as a test to predict these outcomes. DESIGN Multicenter prospective study of in-hospital cardiac arrest. SETTING Five medical centers in the United States and the United Kingdom. PATIENTS Inclusion criteria are as follows: in-hospital cardiac arrest, age 18 years old or older, and prolonged cardiopulmonary resuscitation greater than or equal to 5 minutes. Patients were recruited consecutively during working hours between August 2011 and September 2014. Survival with a favorable neurologic outcome was defined as a cerebral performance category 1-2. INTERVENTIONS Cerebral oximetry monitoring. MEASUREMENTS AND MAIN RESULTS Among 504 in-hospital cardiac arrest events, 183 (36%) met inclusion criteria. Overall, 62 of 183 (33.9%) achieved return of spontaneous circulation, whereas 13 of 183 (7.1%) achieved cerebral performance category 1-2 at discharge. Higher mean ± SD regional cerebral oxygenation was associated with return of spontaneous circulation versus no return of spontaneous circulation (51.8% ± 11.2% vs 40.9% ± 12.3%) and cerebral performance category 1-2 versus cerebral performance category 3-5 (56.1% ± 10.0% vs 43.8% ± 12.8%) (both p < 0.001). Mean regional cerebral oxygenation during the last 5 minutes of cardiopulmonary resuscitation best predicted the return of spontaneous circulation (area under the curve, 0.76; 95% CI, 0.69-0.83); regional cerebral oxygenation greater than or equal to 25% provided 100% sensitivity (95% CI, 94-100) and 100% negative predictive value (95% CI, 79-100); regional cerebral oxygenation greater than or equal to 65% provided 99% specificity (95% CI, 95-100) and 93% positive predictive value (95% CI, 66-100) for return of spontaneous circulation. Time with regional cerebral oxygenation greater than 50% during cardiopulmonary resuscitation best predicted cerebral performance category 1-2 (area under the curve, 0.79; 95% CI, 0.70-0.88). Specifically, greater than or equal to 60% cardiopulmonary resuscitation time with regional cerebral oxygenation greater than 50% provided 77% sensitivity (95% CI,:46-95), 72% specificity (95% CI, 65-79), and 98% negative predictive value (95% CI, 93-100) for cerebral performance category 1-2. CONCLUSIONS Cerebral oximetry allows real-time, noninvasive cerebral oxygenation monitoring during cardiopulmonary resuscitation. Higher cerebral oxygenation during cardiopulmonary resuscitation is associated with return of spontaneous circulation and neurologically favorable survival to hospital discharge. Achieving higher regional cerebral oxygenation during resuscitation may optimize the chances of cardiac arrest favorable outcomes

Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres

BACKGROUND: Controlled human malaria infection (CHMI) accelerates development of anti-malarial interventions. So far, CHMI is done by exposure of volunteers to bites of five mosquitoes carrying Plasmodium falciparum sporozoites (PfSPZ), a technique available in only a few centres worldwide. Mosquito-mediated CHMI is logistically complex, exact PfSPZ dosage is impossible and live mosquito-based interventions are not suitable for further clinical development. METHODS: An open-labelled, randomized, dose-finding study in 18-45 year old, healthy, malaria-naive volunteers was performed to assess if intravenous (IV) injection of 50 to 3,200 aseptic, purified, cryopreserved PfSPZ is safe and achieves infection kinetics comparable to published data of mosquito-mediated CHMI. An independent study site verified the fully infectious dose using direct venous inoculation of PfSPZ. Parasite kinetics were assessed by thick blood smear microscopy and quantitative real time PCR. RESULTS: IV inoculation with 50, 200, 800, or 3,200 PfSPZ led to parasitaemia in 1/3, 1/3, 7/9, and 9/9 volunteers, respectively. The geometric mean pre-patent period (GMPPP) was 11.2 days (range 10.5-12.5) in the 3,200 PfSPZ IV group. Subsequently, six volunteers received 3,200 PfSPZ by direct venous inoculation at an independent investigational site. All six developed parasitaemia (GMPPP: 11.4 days, range: 10.4-12.3). Inoculation of PfSPZ was safe. Infection rate and pre-patent period depended on dose, and injection of 3,200 PfSPZ led to a GMPPP similar to CHMI with five PfSPZ-infected mosquitoes. The infectious dose of PfSPZ predicted dosage of radiation-attenuated PfSPZ required for successful vaccination. CONCLUSIONS: IV inoculation of PfSPZ is safe, well tolerated and highly reproducible. It shall further accelerate development of anti-malarial interventions through standardization and facilitation of CHMI. Beyond this, rational dose selection for whole PfSPZ-based immunization and complex study designs are now possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01624961 and NCT01771848

Formal approaches to number in Slavic and beyond

The goal of this collective monograph is to explore the relationship between the cognitive notion of number and various grammatical devices expressing this concept in natural language with a special focus on Slavic. The book aims at investigating different morphosyntactic and semantic categories including plurality and number-marking, individuation and countability, cumulativity, distributivity and collectivity, numerals, numeral modifiers and classifiers, as well as other quantifiers. It gathers 19 contributions tackling the main themes from different theoretical and methodological perspectives in order to contribute to our understanding of cross-linguistic patterns both in Slavic and non-Slavic languages

Formal approaches to number in Slavic and beyond

The goal of this collective monograph is to explore the relationship between the cognitive notion of number and various grammatical devices expressing this concept in natural language with a special focus on Slavic. The book aims at investigating different morphosyntactic and semantic categories including plurality and number-marking, individuation and countability, cumulativity, distributivity and collectivity, numerals, numeral modifiers and classifiers, as well as other quantifiers. It gathers 19 contributions tackling the main themes from different theoretical and methodological perspectives in order to contribute to our understanding of cross-linguistic patterns both in Slavic and non-Slavic languages