Medical Research Foundation Research Articles

1088. Ultrasensitive Detection of C. difficile Toxins in Stool Using Single Molecule Counting Technology: A Multicenter Study for Evaluation of Clinical Performance

BackgroundCommercially available tests for Clostridium difficile infection (CDI) make test selection by the laboratory difficult due to the following unsatisfactory characteristics: long turnaround time, poor sensitivity, and/or poor specificity. The Singulex Clarity® C. diff toxins A/B assay (in development) is a rapid and automated immunoassay for the detection of C. difficile toxins A and B in stool, with analytical limits of detection for toxins A and B at 2.0 and 0.7 pg/mL, respectively. In this multicenter study, the clinical performance of the Singulex Clarity C. diff toxins A/B assay was compared with standalone PCR, a multistep algorithm with enzyme immunoassay (EIA) and PCR, and cell cytotoxicity neutralization assay (CCNA).MethodsFresh samples from 267 subjects with suspected CDI were tested at two sites (Minneapolis Medical Research Foundation and TriCore Reference Laboratories) with the Singulex Clarity assay, PCR (Xpert®C. difficile), and EIA (C. Diff Quik Chek Complete®) for GDH and toxin testing. The performance of the assays and multistep algorithms were evaluated against CCNA (Microbiology Specialists, Inc.).ResultsThe overall CDI prevalence was 15.7%. The Singulex Clarity C. diff toxins A/B assay had 90.5% sensitivity and 96.0% specificity, with a 98.2% negative predictive value when compared with CCNA, and the Clarity assay’s AuROC was 0.9534. PCR had 90.5% sensitivity and 91.1% specificity. Compared with CCNA, the toxin EIA had 47.6% sensitivity and 100% specificity. Testing with a multistep algorithm using EIA with discordant results reflexed to PCR resulted in 85.7% sensitivity and 94.7% specificity.ConclusionThe ultrasensitive Singulex Clarity C. diff toxins A/B assay is equivalent to the sensitivity of PCR while providing higher specificity. Compared with a multistep algorithm, the Clarity assay provides higher sensitivity and specificity while providing faster time-to-result in a simpler-to-understand, one-step reporting structure, allowing for a standalone, single-step solution for detection of C. difficile toxins in patients with suspected CDI.Disclosures E. Friedland, Singulex, Inc.: Employee, Salary. A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. S. Biscocho, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

Human Bone Marrow-Derived Mesenchymal Stem Cells Promote the Growth and Drug-Resistance of Diffuse Large B-Cell Lymphoma by Secreting IL-6 and Elevating IL-17A Levels

Background: The drug-resistance and relapse of diffuse large B-cell lymphoma (DLBCL), which are related to mesenchymal stem cells (MSCs), have become increasingly common. However, the underlying mechanisms remain elusive. Methods: CCK 8 assay, colony formation assay, and xenograft mouse model were used to investigate the effects of hBMSCs on DLBCL growth.Immunohistochemistry, qRT-PCR, and ELISA were used to study the expressions of IL-6 and IL-17A. Flow cytometry was used to analyze Th17 cells and Treg cells expressions. Western blot analysis, microarray analysis, and bioinformatics analysis were used to analyze the pathways of IL-6 or IL-17A mediated DLBCL growth. Findings: HBMSCs promoted DLBCL growth by secreting IL-6 in vitro and in vivo and simultaneously upregulating IL-17A in vitro. IL-6 and IL-17A synergistically promoted the growth and drug-resistance of DLBCL cells by protecting them from spontaneous or drug-induced apoptosis in vitro. IL-6 or IL-17A activated the JAK2/STAT3 pathway or upregulated cyclin D2 via activation of PI3K/Akt signaling in vitro, respectively. Interpretation: The present results indicated that hBMSCs might have a dual effect on promoting DLBCL progression and drug-resistance by secreting IL-6 and upregulating IL-17A. IL-6, IL-17A, p-STAT3, p-Akt or cyclin D2 may be potential molecular targets for overcoming drug-resistance in patients with relapsed or refractory DLBCL. Funding Statement: This work was supported by the Natural Science Foundation of Guangdong Province, China (2018A0303130167); the Guangzhou Planned Project of Science and Technology, China (201707010279, 201300000100, and 201704020105); the Guangdong Medical Science and Research Foundation, China (A2018258); the Guangzhou General Science and Technology Project of Health and Family Planning, China (20181A011011). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This research was approved by the Ethics Committee of Guangzhou First People’s Hospital (K-2017-066-02). Written informed consent was obtained from all participants or their families. All animal experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at Guangzhou First People's Hospital, Guangzhou Medical University.

Deconvolving the input to random abstract parabolic systems: a population model-based approach to estimating blood/breath alcohol concentration from transdermal alcohol biosensor data**This research was supported in part by grants from the Alcoholic Beverage Medical Research Foundation and the National Institute on Alcohol Abuse and Alcoholism (R21AA017711 and R01AA026368, S.E.L. and I.G.R.), and (R01AA025969, C.E.F.).

The distribution of random parameters in, and the input signal to, a distributed parameter model with unbounded input and output operators for the transdermal transport of ethanol are estimated. The model takes the form of a diffusion equation with the input, which is on the boundary of the domain, being the blood or breath alcohol concentration (BAC/BrAC), and the output, also on the boundary, being the transdermal alcohol concentration (TAC). Our approach is based on the reformulation of the underlying dynamical system in such a way that the random parameters are treated as additional spatial variables. When the distribution to be estimated is assumed to be defined in terms of a joint density, estimating the distribution is equivalent to estimating a functional diffusivity in a multi-dimensional diffusion equation. The resulting system is referred to as a population model, and well-established finite dimensional approximation schemes, functional analytic based convergence arguments, optimization techniques, and computational methods can be used to fit it to population data and to analyze the resulting fit. Once the forward population model has been identified or trained based on a sample from the population, the resulting distribution can then be used to deconvolve the BAC/BrAC input signal from the biosensor observed TAC output signal formulated as either a quadratic programming or linear quadratic tracking problem. In addition, our approach allows for the direct computation of corresponding credible bands without simulation. We use our technique to estimate bivariate normal distributions and deconvolve BAC/BrAC from TAC based on data from a population that consists of multiple drinking episodes from a single subject and a population consisting of single drinking episodes from multiple subjects.

Liver transplantation and waitlist mortality for HCC and non-HCC candidates following the 2015 HCC exception policy change.

Historically, exception points for hepatocellular carcinoma (HCC) led to higher transplant rates and lower waitlist mortality for HCC candidates compared to non-HCC candidates. As of October 2015, HCC candidates must wait 6months after initial application to obtain exception points; the impact of this policy remains unstudied. Using 2013-2017 SRTR data, we identified 39 350 adult, first-time, active waitlist candidates and compared deceased donor liver transplant (DDLT) rates and waitlist mortality/dropout for HCC versus non-HCC candidates before (October 8, 2013-October 7, 2015, prepolicy) and after (October 8, 2015-October 7, 2017, postpolicy) the policy change using Cox and competing risks regression, respectively. Compared to non-HCC candidates with the same calculated MELD, HCC candidates had a 3.6-fold higher rate of DDLT prepolicy (aHR=3.49 3.69 3.89 ) and a 2.2-fold higher rate of DDLT postpolicy (aHR=2.09 2.21 2.34 ). Compared to non-HCC candidates with the same allocation priority, HCC candidates had a 37% lower risk of waitlist mortality/dropout prepolicy (asHR=0.54 0.63 0.73 ) and a comparable risk of mortality/dropout postpolicy (asHR=0.81 0.95 1.11 ). Following the policy change, the DDLT advantage for HCC candidates remained, albeit dramatically attenuated, without any substantial increase in waitlist mortality/dropout. In the context of sickest-first liver allocation, the revised policy seems to have established allocation equity for HCC and non-HCC candidates.

Impact of the health education and preventive equipment package (HEPEP) on prevention of Strongyloides stercoralis infection among rural communities in Northeast Thailand: a cluster randomized controlled trial

BackgroundStrongyloidiasis is prevalent in northeast Thailand. This study aimed to evaluate the impact of the Health Education and Preventive Equipment Package (HEPEP), a package we developed to improve awareness and aid in the prevention of Strongyloides stercoralis infection among rural communities in northeast Thailand.MethodsThis was an intervention trial conducted in 12 villages (six interventions and six controls) in rural areas of northeast Thailand from March 2016 to September 2017. Single stool sample was collected from each participant and examined using agar plate culture (APC) technique. Each participant was interviewed using a pre-tested questionnaire, treated with single dose of ivermectin (200 μg/Kg), and allocated to either the intervention or control group. Members of the intervention group were given “Practices to Prevent Strongyloidiasis” poster and vinyl boards containing information aimed at raising awareness of S. stercoralis and strongyloidiasis. In addition, they were given a poster lecture regarding the lifecycle of S. stercoralis before being treated with ivermectin. Aside from that, they were also given a protective equipment package. Monthly refresher courses were provided by village health volunteers (VHVs) regarding the health information they had received and proper equipment usage. The control group, on the other hand, was only provided with a five-minute lecture regarding strongyloidiasis. Assessment of new infection was conducted 3 months later in 327 and 318 participants in the intervention group and control group, respectively.ResultsThe HEPEP had 41% greater efficacy in preventing S. stercoralis infection in the intervention group than the measures taken in the control group (adjusted Odds Ratio (aOR) = 0.59; 95%CI: 0.41 to 0.85, P-value = 0.005). The intervention group also scored significantly higher on all aspects of a test of S. stercoralis knowledge compared with the control group (mean difference (mean dif.) = 2.89, P-value = < 0.05).ConclusionsThe HEPEP was the first model that has been found to be effective in controlling of S. stercoralis in rural communities in the northeast Thailand. The results should encourage policy makers and public health personnel to improve control programs, as well as health promotion, with regard to parasites.Trial registrationThai Clinical Trials Registry (TCTR), Medical Research Foundation of Thailand, Medical Research Network of the Consortium of Thai Medical Schools: MedResNet (Thailand) (identification number: TCTR20180404002) Registered 4 April 2018 (retrospectively registered).

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study

SummaryBackgroundHuman genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress.MethodsWe did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria.FindingsBetween June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002).InterpretationBoth ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health.FundingWellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Who can tolerate a marginal kidney? Predicting survival after deceased donor kidney transplant by donor-recipient combination.

The impact of donor quality on post-kidney transplant (KT) survival may vary by candidate condition. Characterizing this variation would increase access to KT without sacrificing outcomes. We developed a tool to estimate post-KT survival for combinations of donor quality and candidate condition. We studied deceased donor KT recipients (n=120818) and waitlisted candidates (n=376272) between 2005 and 2016 by using the Scientific Registry of Transplant Recipients. Donor quality and candidate condition were measured by using the Kidney Donor Profile Index (KDPI) and the Estimated Post Transplant Survival (EPTS) score. We estimated 5-year post-KT survival based on combinations of KDPI and EPTS score using random forest algorithms and waitlist survival by EPTS score using Weibull regressions. Survival benefit was defined as absolute reduction in mortality risk with KT. For candidates with an EPTSscore of80, 5-year waitlist survival was 47.6%, and 5-year post-KT survival was 78.9% after receiving kidneys with a KDPIof20 and was 70.7% after receiving kidneys with a KDPIof80. The impact of KDPI on survival benefit varied greatly by EPTS score. For candidates with low EPTS scores (eg, <40), the KDPI had limited impact on survival benefit. For candidates with middle or high EPTS scores (eg, >40), survival benefit decreased with higher KDPI but was still substantial even with a KDPIof100 (>16 percentage points). Our prediction tool (www.transplantmodels.com/kdpi-epts) can support individualized decision-making on kidney offers in clinical practice.

Abstract 4763: NCI's Expert-Vetted Biospecimen Evidence-Based Practices (BEBP): A resource for harmonization of biospecimen collection procedures

Abstract High quality human biospecimens are essential to clinical diagnosis and treatment and serve as a vital foundation for medical research. Substantial variation in procedures for biospecimen collection, processing, and storage exists within and between institutions. Such variation can have a substantial effect on the quality of biospecimens and the accuracy of downstream analytical results, and is of particular concern at a time of high demand for biospecimens for large-scale studies. Recognizing the need for harmonization of biospecimen practices, the National Cancer Institute (NCI)'s Biorepositories and Biospecimen Research Branch (BBRB) began developing a series of documents known as Biospecimen Evidence-Based Practices (BEBPs). The first of NCI's BEBPs, “Snap-Freezing of Post-surgical Tissue Biospecimens” (https://biospecimens.cancer.gov/global/pdfs/NCI_BEBP_Snap-freezing_of_Post-surgical_Tissue_Biospecimens.pdf), was released in 2014. BBRB will soon release two new expert-reviewed BEBPs. The first expert-vetted BEBP, “Formalin-Fixation and Paraffin Processing of Tissue Biospecimens”, is a fit-for-purpose guide for formalin fixation, paraffin impregnation, and embedding of human tissues that was reviewed by more than 40 experts as part of a BBRB-sponsored workshop. The second, entitled “DNA and RNA Extraction from Formalin-fixed Paraffin-embedded Tissue Biospecimens”, categorizes steps in the extraction of DNA and RNA that may affect the quality of nucleic acids obtained from FFPE specimens and was reviewed by five experts. Each BEBP has been developed in close partnership with scientists having state-of-the art biospecimen science expertise and a strong publication record. BEBP documents contain step-by-step recommendations and procedural guidelines as well as a summary of supporting literature and expert-derived evidence. Development of each BEBP employs a stringent and thorough analysis of literature indexed in the National Library of Medicine's PubMed database and curated in NCI's publicly accessible Biospecimen Research Database (BRD; http://biospecimens.cancer.gov/brd), a free database of peer-reviewed biospecimen science articles. The database also houses over 400 SOPs searchable by numerous parameters including specimen type, preservation, diagnosis, analyte, pre-analytical factors, and technology platform. Given the nuanced requirements for each individual step in biospecimen collection, NCI's BEBPs serve as a framework to inform the development of Standard Operating Procedures (SOPs) by individual institutions while providing room for flexibility and adaptation. NCI's BBRB continues to add to its BEBP series to improve the reproducibility of molecular data derived from biospecimens. Experts interested in contributing to future BEBPs are encouraged to contact BBRB at ncibbrb@nih.gov. Citation Format: Esmeralda Casas-Silva, Lori D. Campbell, Kelly Engel, Sarah Greytak, Ping Guan, Helen Moore. NCI's Expert-Vetted Biospecimen Evidence-Based Practices (BEBP): A resource for harmonization of biospecimen collection procedures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4763.

Comparable Levels of Inflammatory Mediators in Portal Venous Blood Collected from Organ Donors Donating after Circulatory Death and those Donating after Brain Death

Introduction Extended criteria donors, such as those who donate after cardiac death (DCD) are increasingly considered for transplantation. Biliary stricture formation often complicates the use of these grafts and the aetiology remains largely unknown. We have previously shown that endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However, it remains unclear if endotoxaemia occurs during DCD organ donation. The aim of this study was to determine the inflammatory propensity of portal blood collected from DCD donors compared to those donating after brain death (DBD). Materials and Methods Serial portal venous as well as hepatic venous blood samples were collected from adult DBD and DCD organ donors. An NF-κB-dependent cell-based assay was used to detect endotoxins and/or other inflammatory stimuli in the portal blood samples. β-galactosidase activity in hepatic venous samples was assessed as a marker for hepatic Kupffer cell activation. In addition, bile and bile duct tissue was collection for assessment of biliary injury. Results and Discussion Thirty patients (nine DCD, 21 DBD) were included in this study. Compared to DBD donors, portal samples of DCD donors did not have an enhanced propensity for triggering inflammatory responses. However, Kupffer cell activation was enhanced and prolonged in DCD donors. Lactate dehydrogenase as a biomarker of biliary injury was increased in bile collected from DCD donors and histological scoring showed evidence of increased injury of peri-luminal peribiliary glands. Conclusion In this study no evidence was found of endotoxaemia or increased propensity to produce an inflammatory response in portal blood collected from DCD donors. Despite this, hepatic Kupffer cell activation was increased and there was evidence of biliary injury. Liver Transplant Bequest, PA Research Foundation, Princess Alexandra Hospital, Brisbane, Australia. Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Brisbane, Australia.

Abstract 5083: Using proteomics profiling to elucidate the interactions of breast cancer-derived exosomes with the blood-brain barrier

Abstract Breast cancer metastasis to brain is associated with a dismal prognosis commonly attributed to a limited understating of the mechanisms driving this pathological process. Elucidation of the early events leading to brain metastasis is essential to the development of more effective therapeutic and diagnostic approaches. With an interest in the role of breast cancer-derived exosomes in brain metastasis, our group has previously shown that exosomes derived from a brain-seeking variant of the breast cancer cell line MDA-MB-231 (Br-Ex) can facilitate brain metastasis by inducing alterations in the protein expression profile of astrocytes, one of the components of the blood brain barrier (BBB). This observation led us to hypothesize that the interaction(s) between exosomes and astrocytes is more efficient compared to brain endothelial cells (ECs) or pericytes, the two additional major components of the BBB, resulting in more prominent alterations in the protein expression profile of astrocytes. To test this hypothesis, we first quantified and compared the uptake of exosomes by brain ECs, astrocytes, and pericytes in vitro. The uptake of Br-Ex by astrocytes was significantly greater than that of brain ECs (P&amp;lt;4e-3) and pericytes (P&amp;lt;1e-3). In contrast, exosomes derived from parental or bone-seeking MDA-MB-231 cells (P-Ex and Bo-Ex, respectively) did not show a preferential uptake by astrocytes. We have also demonstrated the uptake of Br-Ex by astrocytes in vivo. The uptake of exosomes by different cell types predominantly relies on the interaction of exosomal proteins with different receptors on the recipient cells. To determine the exosomal proteins potentially involved in the preferential uptake of Br-Ex by astrocytes, we performed quantitative mass spectrometry on the P-, Bo-, and Br-Ex via Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) analysis. Database searches were performed against human proteins in the SwissProt database and a total of 126 proteins were detected with over 95% confidence. Pairwise comparisons identified a total of 27 and 21 proteins with statistically significant differential expression (P &amp;lt; 0.05) in the Br-Ex compared to the P- and Bo-Ex, respectively. Functional enrichment analysis of the detected proteins demonstrated that proteins belonging to the cell migration and focal adhesion categories were over-represented in the Br-Ex. Among these proteins, a number of integrins and annexins were highly enriched in the Br-Ex and can potentially be involved in the preferential uptake of these exosomes by astrocytes. These findings indicate that exosomes derived from brain-seeking breast cancer cells can preferentially interact with astrocytes and these interactions can be driven by exosomal integrins and annexins. (The authors are grateful for the support of the Breast Cancer Research Foundation and the Advanced Medical Research Foundation.) Citation Format: Golnaz Morad, Hasan H. Otu, Simon T. Dillon, Marsha A. Moses. Using proteomics profiling to elucidate the interactions of breast cancer-derived exosomes with the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5083.

Outcome of neonates born to mothers with premature rupture of membranes

Background: Premature rupture of membranes (PROM) is defined as rupture of membranes before the onset of labor which is caused by maternal intrauterine infection which increases with the duration of membrane rupture seen in one third cases of all preterm birth. The primary complication for the mother is risk of infection, complications for the newborn consists of prematurity, foetal distress, cord compression, deformation and altered pulmonary development. The present study was undertaken to evaluate the outcome of neonates born to mothers with PROM and know the incidence of adverse outcomes in neonates and the incidence of early onset sepsis following PROM more than 18hours in mothers who have not received intrapartum antibiotics.Methods: Prospective cohort study conducted in between December 2015 to November 2017 in which 100 neonates born to mothers with history of Premature rupture of membranes admitted in Konaseema Institute and Medical Science and Research Foundation, Amalapuram were selected for this study. They were evaluated with various investigations by a written proforma and outcomes of these neonates were studied.Results: Out of 100 cases of PROM 54 had 18-24 hours duration, 38 had 24-72hours and 8 had more than 72 hours duration of PROM. According to swab culture organism found were E Coli in 22, Staphylococcus in 20, Klebsiella in 12, Pseudomonas in 8 cases with rest 38 cases showed no growth. 49 out of 100 cases show morbidity with Respiratory distress being the most common with 37 cases followed by septicaemia with 8 cases. Morbidity are more in the neonate with longer duration (&gt;24hrs) of PROM. Mortality increases progressively as the duration of PROM increases.Conclusions: In neonates born after PROM of &gt;24 hours, risk of maternal infection, neonatal morbidity and mortality is more compared to those with PROM of shorter duration. Active management is needed to enable delivery within 24 hrs of premature rupture of membranes as it is associated with better neonatal outcome compared to longer latency period.

At BIO, business boomed, and drug pricing loomed

Some 17,000 people descended on Boston during the first week of June for the annual meeting of the Biotechnology Innovation Organization (BIO). If the crowds were any indication, business is booming. “This has been by far the most successful BIO convention we’ve ever had,” said John Maraganore, CEO of Alnylam Pharmaceuticals and chair of BIO. Attendees set a Guinness world record for the largest business-partnering event, with more than 45,000 meetings among people from across the globe. “If you want to be in the business of biotechnology, you need to be here,” said Manu Nair, who came to Boston from the Oklahoma Medical Research Foundation. Biotech investment is on track for a record year. “The first quarter was very strong for venture capital investing, and those trends seem durable,” said Glen Giovannetti, global biotechnology leader at the consulting firm EY. Cancer companies, especially immuno-oncology start-ups, are attracting a disproportionate amount

Study on the knowledge of pharmacovigilance among medical students

Background: Pharmacovigilance is the science and activity relating to the detection, assessment, understanding and prevention of adverse effects or any other drug–related problems. The ultimate aim of pharmacovigilance is to ensure safe and rational use of medicines, once they are released for general use in the society. The most important outcome of pharmacovigilance is the prevention of negative consequences of pharmacotherapy. So, the present study primary objective is to assess awareness of pharmacovigilance among medical students and to evaluate the knowledge of pharmacovigilance at Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation, Chinaoutpally (A.P.), a tertiary care teaching hospital.Methods: A cross-sectional study will be carried out using a pretested questionnaire. The questionnaire will be designed to assess the knowledge regarding pharmacovigilance. The medical students (Final MBBS and Interns) were included in the study during the study period. Only the participants who will give their consent will be included in the study. Results will be calculated by proper statistical analysis.Results: The current study was conducted in a medical college Dr. PSIMS and RF, which included a total of 180 medical students 75% final years, 25% interns. Most of them (89.8%) accepted that reporting ADR is necessary and pharmacovigilance should be taught in detail to healthcare professionals.Conclusions: This study demonstrated that knowledge of pharmacovigilance among medical students is improving gradually, but unfortunately the actual practice of ADR reporting is still deficient among students.

Does otitis media in early childhood affect later behavioural development? Results from the Western Australian Pregnancy Cohort (Raine) Study.

To examine the relationship between early life episodes of otitis media and later behavioural development with adjustment for confounders. Longitudinal cohort study. The Western Australian Pregnancy Cohort (Raine) Study recruited 2900 pregnant women from King Edward Memorial Hospital (KEMH) in Perth, Western Australia, between 1989 and 1991. Data from the children born were collected at both the Year 3 and Year 5 follow-up. At Year 3, n=611 were diagnosed with recurrent otitis media through parent-report and clinical examination. At Year 5, n=299 were considered exposed to otitis media based upon tympanometry results. Performance in the Child Behaviour Checklist (CBCL), a questionnaire completed by the primary caregiver at Year 10. Significant associations were found between recurrent otitis media at Year 3 and internalising behaviours (P=.011), and the somatic (P=.011), withdrawn (P=.014), attention (P=.003) and thought problems domains (P=.021), and the total CBCL score (P=.010). A significant association was also found between exposure to otitis media at Year 5 and externalising behaviours (P=.026). A modest association was seen between recurrent otitis media at Year 3 and exposure to otitis media at Year 5 and a number of behaviour domains at Year 10.

Twin Talk Series: A novel method to foster peer‐to‐peer pedagogical training

Mayo Clinic annually trains over 1000 predoctoral students and postdoctoral research fellows in medicine and biomedical sciences. However, training opportunities are limited for those interested in teaching and mentoring methodologies. Therefore, Mayo Clinic predoctoral students sought to provide trainee‐led learning opportunities for fellow trainees. A survey was sent to all Mayo Clinic predoctoral and postdoctoral trainees to determine preferences for pedagogy education formats. In response to survey results, the Twin Talk Series was developed. A Twin Talk is a set of one‐hour talks comprised of a Take‐in session and one or more Try‐out session(s). At the Take‐in session, a content expert discusses a pedagogical technique, and at the following Try‐out session(s), trainee volunteers practice the technique among themselves. Eight Twin Talks have been organized to date on several teaching techniques and pedagogical development topics. The impact of each Twin Talk, including understanding, format, and interest level, was evaluated through survey tools. Survey results suggested trainees favored in‐person pedagogy expert presenters during the Take‐in session over video conferences. Furthermore, modeling of the teaching technique by the expert presenter during the Take‐in session was well‐received. However, participation in the follow‐up Try‐out practice session was limited. Overall, the Twin Talk Series is a promising method for trainee‐led, peer‐to‐peer pedagogy education, particularly at institutions with limited access to pedagogical resources. The Twin Talk Series will continue to be refined to increase trainee participation, particularly in the follow‐up Try‐out sessions, and to provide relevant opportunities for trainees to practice teaching.Support or Funding InformationMayo Foundation of Medical Education and ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Early blood pressure response to isometric exercise is attenuated in obese individuals who have undergone bariatric surgery

ObjectiveBlood pressure (BP) reactivity to stress is predictive of the development of cardiovascular disease. Cardiovascular reactivity is attenuated in individuals after bariatric surgery; however, the underlying mechanisms are not completely understood. We hypothesized the BP response at the onset of isometric handgrip exercise would occur earlier and to a lesser degree in individuals who underwent bariatric surgery when compared to obese adults and the reliance on total peripheral resistance (TPR) would be attenuated.MethodsTwenty‐six individuals [7 non‐obese (28±3 yrs, 23±1 kg/m2), 11 obese (33±2 yrs, 39±1 kg/m2), 8 post‐bariatric surgery (35±2 yrs, 29±1 kg/m2)] completed isometric handgrip exercise (40% maximum voluntary contraction) to exhaustion. Heart rate (HR, ECG) and arterial BP (brachial catheter) were measured continuously. Stroke volume was estimated from the pressure waveform and cardiac output (CO) and TPR were calculated. Peak change, time to peak, and rate of rise in BP were assessed during the first 30‐s of exercise.ResultsCompared to non‐obese controls, obese adults exhibited a slower initial rise in BP (9.3± 3.9 vs 21.7±2.5 s) and higher peak BP change (Δ6.7±2.1 vs Δ11.9±2.1 mmHg) at the onset of isometric handgrip exercise (p&lt;0.05). Peak BP and the rate of rise were not different between individuals who underwent bariatric surgery (14.0±4.1 s, Δ5.6±1.3 mmHg) and non‐obese controls (p&gt;0.05). Non‐obese controls exhibited an exercise‐mediated increase in CO whereas obese adults increased TPR (p&lt;0.05). The increases in CO and TPR were less apparent in individuals who underwent bariatric surgery (p&gt;0.05). Low cardiac baroreflex sensitivity (BRS) and heart rate variability (HRV: SDNN, standard deviation of normal to normal R‐R intervals) at rest were related to smaller increases in CO (BRS: R=0.47, p&lt;0.01; SDNN: R=0.57, p&lt;0.01) and greater increases in TPR (BRS: R=−0.49, p=0.01; SDNN: R=−0.60, p&lt;0.01) during exercise.ConclusionsIn contrast to obese adults, individuals who underwent bariatric surgery exhibit a rapid rise in BP at the onset of isometric handgrip exercise. This rapid increase in BP is associated with a fall in TPR and results in lower peak BP at the onset of isometric exercise. These data suggest bariatric surgery attenuates BP reactivity to physical stress via improvements in the time‐course of hemodynamic responses and reflex autonomic adjustments.Support or Funding InformationNIH HL130339, NIH HL083947, NIH DK82424, NIH UL1 RR024150, Mayo Clinic Department of Anesthesiology, Mayo Foundation for Medical Education and ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organelle Segregation and Quality Control during Meiotic Differentiation

Meiotic differentiation is a complex developmental program that involves extensive cellular remodeling. In budding yeast, under nutrient deprived conditions, a diploid mother cell undergoes two successive meiotic divisions to generate four nuclei. After meiosis, the four nuclei are packaged with a subset of the organelles and cytoplasm originating from the mother to form four haploid gametes. Our lab has found that during the latter process, certain components of the mother cell are not inherited by the gametes and are subsequently produced de novo. For example, the nuclear pore complexes and components of the nucleoli do not segregate with any of the four nuclei, whereas only a subset of the mitochondria is inherited by the gametes. A programmed pathway that entails vacuole permeabilization subsequently degrades the excluded cellular materials. We posit that the restricted inheritance of organelles and other cellular sub‐compartments is required for cellular rejuvenation that occurs as part of gametogenesis.Support or Funding InformationPew Charitable Trusts (00027344), Damon Runyon Cancer Research Foundation (35‐15), March of Dimes (5‐FY15‐99), National Institute of Health (DP2 AG055946‐01) and Glenn Foundation for Medical ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Microprotein‐Protein Interactions via APEX Tagging

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Genomics and proteomics technologies have led to the recent discovery of hundreds to thousands of new microproteins. Emerging evidence demonstrates that these microproteins have fundamental roles in various biological processes including metabolism, apoptosis, and development, highlighting the value of characterizing these molecules. The identification of microprotein‐protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of non‐specific interactions for microproteins. To address this issue, we tested and applied an in situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The results demonstrated that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX‐tagging to two uncharacterized microproteins, C11orf98‐MP and MIEF‐MP revealed that they interact with nucleolar proteins nucleophosmin and nucleolin (C11orf98‐MP) and acyl carrier protein in mitochondria (MIEF‐MP) respectively, demonstrating the ability of this approach to identify novel hypothesis‐generating MPIs.Support or Funding InformationQ.C. is a postdoctoral fellow funded by the George E. Hewitt Foundation for medical research. This study was supported by the NIH with an NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.) and R01 (GM102491, A.S.), the Leona M. and Harry B. Helmsley Charitable Trust grant (#2012‐PG‐MED002, A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.). Imaging work in this study was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NINDS Neuroscience Core Grant: NS072031 and the Waitt Foundation. We also want to thank the Cancer Center Support Grant P30 CA014195 for purchasing and providing access to the MGC collection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activator Protein 1 and Caspase 8 Mediate Palmitate‐Induced Cardiomyocyte Apoptosis

IntroductionLipoapoptosisof cardiomyocytes may be the basis for diabetic cardiomyopathy. Wepreviously demonstrated that p38α mitogen activated protein kinase (MAPK) mediatespalmitate (PA)‐induced cardiomyocyte apoptosis, and that specific p38α siRNAmarkedly and dose‐dependently attenuates apoptosis in cardiomyocytes treated with PA. We tested the hypothesis that p38α‐dependent pathway consists, in part, of c‐fos subunit of Activator Protein 1 (AP‐1) and caspase8, and that this p38α/AP‐1/caspase 8 cascade mediate PA‐induced cardiomyocyte apoptosis.MethodsHuman adult ventricular cardiomyocytes line (AC16 cells) were exposed to high physiological levels of PA. p38α‐dependent pathway was evaluated using p38α and c‐fos siRNA knockdown, and caspase 8 inhibitor.ResultsWith PA treatment, dose‐dependent increases in c‐fos mRNA (1.83 fold and 3.13 fold control, at 150 μM and 300 μM respectively, n=6, p&lt;0.01), in c‐fos protein (1.00 fold and 2.67 fold, at 150 μM and 300 μM respectively, n=3, p&lt;0.05), and a much greater increase in phospho‐c‐fos (2.64 fold and 8.94 fold, at 150 μM and 300 μM respectively, n=, p&lt;0.01) were measured. Transcription of caspase 8 also increased (1.40 fold and 1.80 fold, at 150 μM and 300 μM respectively, n=5, p&lt;0.05) but procaspase 8 protein level did not.p38α knockdown dose‐dependently blocked the PA‐induced increase in phospho‐c‐fos protein level(to 62%, 45%, and 30% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmol si‐p38α knockdown groups, n=5, p&lt;0.01), but total c‐fos level did not change significantly. Interestingly, procaspase 8 level was also dose‐dependently reduced by p38α knockdown (75%, 65%, and 59% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmolsi‐p38α knockdown groups, n=5, p&lt;0.05).With c‐fos knockdown, PA‐induced apoptosis was reduced 39% (n=3, p&lt;0.05) 24 h post‐transfection, and by 53% after 48 h post‐transfection (n=3, p&lt;0.01). Caspase 8 inhibition for 24 h reduced apoptosis by 39% (n=3, p&lt;0.01).ConclusionsThis study in cardiomyocytes demonstrated that PA increases expression and phosphorylation of c‐fos subunit of AP‐1, and caspase 8 transcription, and further that p38α knockdown attenuates PA‐induced increases in c‐fos and phospho‐c‐fos, and decreases caspase 8. Translational knockdown of c‐fos attenuated PA‐induced cardiomyocyte apoptosis, as did inhibition of caspase 8. Our results support the hypothesis that PA‐induced apoptosis is mediated by p38α, AP‐1/c‐fos, and caspase8.Support or Funding InformationThis work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&amp;D5I01CX000598grant (to P.R.), and VA Merit grant BLRDI01BX007080(to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. The contents and the views do not represent the views of the Department of Veterans Affairs or the US government.

Spironolactone Prevents Elevated Expression of Vascular ENaC in Hypertension

Epithelial sodium channel (ENaC) in the kidneys plays an important role in maintaining salt/water homeostasis and blood pressure. Recent research has demonstrated expression of ENaC in the vasculature, and a new role in regulation of the vascular tone. An increase in vascular tone is seen in vascular diseases such as hypertension indicating that changes in ENaC expression in the vasculature may contribute to hypertension. In this study, we hypothesized ENaC expression in arteries will increase in hypertension and that the treatment with antihypertensive drug, spironolactone (spiro‐) will reduce ENaC expression in arteries.Conduit (aorta) and resistance (mesenteric) arteries from male transgenic rats (Cyp1a1‐ren2) with diet‐inducible hypertension (0.167% indole‐3‐carbinol) were isolated and a‐, b‐ and g‐ENaC expression was analyzed by qRT‐PCR. Arteries from four animal groups were analyzed: (1) normotensive (2) hypertensive, (3) normotensive spiro‐ and (4) hypertensive spiro. Rats were fed a control or hypertensive diet for 6 weeks and in two groups (3 &amp; 4) spiro‐ (4.41mg/kg/day) was introduced 2 weeks after the start of the diet.In mesenteric arteries of hypertensive animals a significant increase in both a‐ENaC (p=0.02) and g‐ENaC mRNA level (p=0.04) was observed in comparison to normotensive arteries. In aortas, there was a significant increase in a‐ENaC mRNA level (p=0.006) in hypertension but no change in b‐ and g‐ENaC expression. In mesenteric arteries, spiro‐treatment decreased a‐ENaC and g‐ENaC in comparison to hypertensive controls. In aortas, a‐ENaC mRNA level was significantly decreased (p=0.002) by spiro‐treatment compared to hypertensive controls.In summary, we observed an upregulation of vascular ENaC mRNA in arteries during hypertension and found that antihypertensive treatment with spironolactone decreased this upregulation. This data suggests a strong link between pathogenesis of hypertension and vascular ENaC expression.Support or Funding InformationUniversity of Otago Doctoral Scholarship, Department of Physiology, Otago Medical Research Foundation, Lottery Grants BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.