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The Rivas Cohort Study: design and baseline characteristics of a Nicaraguan cohort

Tue, 08/16/2016 - 12:09pm

BACKGROUND: A lack of advanced healthcare information systems and validated scientific cohorts in Nicaragua makes it difficult to estimate disease prevalences and other public health statistics. Although there is concern of an "epidemic" of chronic kidney disease (CKD) in this country, statistics regarding its magnitude are derived from only a small number of non-representative studies. Budgetary constraints and the logistical problems of maintaining a study cohort make longitudinal studies difficult. The Rivas Cohort was created to measure disease burden of CKD and other public health priorities in the Department of Rivas, Nicaragua. Using primarily volunteer research students and technologic innovation including GPS, digital photography and point of care biochemical analysis, the ability to establish a longitudinal chronic disease cohort is demonstrated.

METHODS: Subjects were recruited from consecutive adjacent households in thirty-two randomly selected communities in the ten municipalities that comprise the Department of Rivas in southern Pacific coastal Nicaragua. The study was conducted in two phases. In the first phase, subjects were enrolled into the cohort and consented for future re-contact. In Phase II, conducted two years later, attempts were made to re-contact 400 of these subjects for additional data collection. Demographic, lifestyle, occupational, exposure and health data was collected for both phases of the study. Blood and urine testing and height, weight and blood pressure measurements were also performed. GPS coordinates of homes were recorded and maps of remote communities created.

RESULTS: Of 1397 adults living in 533 households approached for participation a total of 1242 (89 %) were enrolled in the cohort. The median age is 41 years and 43 % are male, demographics in agreement with Nicaraguan census data for the Department of Rivas. During Phase II we attempted to re-contact 400 subjects for a follow-up study of CKD. It was possible to re-contact 84 % of these participants and of those re-contacted 95 % agreed to participate in the follow-up study. Of subjects that were not successfully re-contacted the majority had either moved (32) or were not at home (22) at the time of the study team visits.

CONCLUSION: The Rivas Cohort Study enrolled a representative sample of 1242 adults living in the Department of Rivas, Nicaragua. The high re-contact and participation rates at two years suggests that the cohort is suitable for long-term studies and presents opportunities for investigations of disease prevalence, incidence, treatment and other public health matters. GPS coordinates and maps are available for future researchers who wish to use the cohort for additional studies.

Factors associated with knowledge of a Good Samaritan Law among young adults who use prescription opioids non-medically

Tue, 08/16/2016 - 12:09pm

BACKGROUND: To date, no studies have examined the extent of knowledge and perceptions of Good Samaritan Laws (GSLs) among young adults who engage in non-medical prescription opioid (NMPO) use. We sought to determine awareness of and factors associated with knowledge of Rhode Island's Good Samaritan Law (RIGSL) among young adult NMPO users.

FINDINGS: We compared the sociodemographic and overdose-related characteristics of participants who were aware and unaware of the RIGSL and determined independent correlates of knowledge of the RIGSL via modified stepwise logistic regression. Among 198 eligible participants, 15.7 % were black, 62.1 % white, and 20.7 % mixed or other race. The mean age was 24.5 (SD = 3.2) and 129 (65.2 %) were male. Fewer than half (45.5 %) were aware of the RIGSL; nonetheless, the majority (95.5 %) reported a willingness to call 911 in the event of an overdose. Knowledge of the RIGSL was associated with older age, white race, a history of incarceration, a history of injection drug use, lifetime heroin use, ever witnessing or experiencing an overdose, having heard of naloxone, knowledge of where to obtain naloxone, and experience administering naloxone (all p < 0.05). In the final explanatory regression model, lifetime injection drug use, having heard of naloxone, and knowledge of where to obtain naloxone were independently associated with awareness of the RIGSL.

CONCLUSIONS: Fewer than half of NMPO users surveyed knew of the RIGSL. Targeted harm reduction education is needed to address a vulnerable population of NMPO users who have not initiated injection drug use and are unaware of naloxone. Additional research is needed to determine how the effectiveness of GSLs could be improved to prevent overdose deaths among young adults.

Microglia contribute to circuit defects in Mecp2 null mice independent of microglia-specific loss of Mecp2 expression

Tue, 08/16/2016 - 12:09pm

Microglia, the resident CNS macrophages, have been implicated in the pathogenesis of Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. However, the mechanism by which microglia contribute to the disorder is unclear and recent data suggest that microglia do not play a causative role. Here, we use the retinogeniculate system to determine if and how microglia contribute to pathogenesis in a RTT mouse model, the Mecp2 null mouse (Mecp2(tm1.1Bird/y)). We demonstrate that microglia contribute to pathogenesis by excessively engulfing, thereby eliminating, presynaptic inputs at end stages of disease ( > /=P56 Mecp2 null mice) concomitant with synapse loss. Furthermore, loss or gain of Mecp2 expression specifically in microglia (Cx3cr1(CreER);Mecp2(fl/y)or Cx3cr1(Cr)(eER); Mecp2(LSL/y)) had little effect on excessive engulfment, synapse loss, or phenotypic abnormalities. Taken together, our data suggest that microglia contribute to end stages of disease by dismantling neural circuits rendered vulnerable by loss of Mecp2 in other CNS cell types.

End Sequence Analysis ToolKit (ESAT) expands the extractable from single cell RNA-seq experiments

Tue, 08/16/2016 - 12:09pm

RNA-seq protocols that focus on transcript termini are well-suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3'-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1,000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified 9 distinct cell types, three distinct beta-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single cell RNA end-sequencing.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia

Tue, 08/16/2016 - 12:09pm

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced beta cell mass, fewer proliferating beta cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from beta cells in mice.

The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L

Tue, 08/16/2016 - 12:09pm

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis.

Levels of physical dependence on tobacco among adolescent smokers in Cyprus

Tue, 08/16/2016 - 12:09pm

PURPOSE: The purpose of this study is to assess tobacco dependence among Cypriot adolescents and examine its association to cigarette consumption and attitudes towards smoking.

METHODS: The current study used cross-sectional data from the 2011 Cyprus Global Youth Tobacco Survey which adopted multistage cluster sampling methods to select adolescents registered in middle and high schools in Cyprus. Tobacco use, physical dependence on tobacco, and attitudes towards tobacco use were measured in 187 adolescents aged 13-18years old who reported that they had smoked at least once in the preceding 30 days. Physical dependence was assessed using the Levels of Physical Dependence scale.

RESULTS: Physical dependence was present in 86% of the adolescent smokers. The mean latency to needing among smokers in the highest dependence group was 101h. Significant associations were observed between physical dependence and the perceived difficulty in quitting (OR=13.1, 95% CI: 4.0, 43.0) as well as the expectation to continue smoking for the next five years (OR=3.3, 95% CI: 1.3, 8.4). Significant associations were also observed between physical dependence and the number of smoking days per month, daily smoking, daily cigarette consumption, lifetime cigarette consumption, and perceived difficulty in abstaining from smoking for one week.

CONCLUSIONS: Physical dependence provides a symptom-based approach to assess dependence and it is a strong predictor of adolescents' perceptions of their ability to quit or to refrain from smoking for a week. Physical dependence on tobacco was highly prevalent among adolescent smokers in Cyprus and it was associated with greater perceived difficulty in quitting. Interventions targeting adolescent smoking must account for the high prevalence of physical dependence.

Single Molecule Visualization of the DEAH-Box ARPase Prp22 Interacting with the Spliceosome: A Dissertation

Mon, 08/15/2016 - 11:28am

In eukaryotes, the spliceosome is a macromolecular ribonucleoprotein machine that excises introns from pre-mRNAs through two sequential transesterification reactions. The chemistry and fidelity of pre-mRNA splicing are dependent upon a series of spliceosomal rearrangements, which are mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily of proteins, whose members couple ATP hydrolysis to promote RNP structural rearrangements within the spliceosome. This is typified by Prp22, which promotes release of the spliced mRNA from the spliceosome and ensures fidelity of the second step of splicing. This role is well documented through classical biochemical and yeast genetics methods. Yet very little is known regarding the comings and goings of Prp22 relative to the spliceosome. My thesis research investigated the dynamics of Prp22 during splicing by using single-molecule fluorescence methods that allowed direct observation of these events. To do this, I helped construct a toolkit that combined yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS) with in vitro splicing assays. Specifically, my thesis research consisted of CoSMoS splicing experiments in which fluorescently labeled pre-mRNA, spliceosome components and Prp22 were directly visualized and analyzed. Using these methods, I found that Prp22’s interactions with the spliceosome are highly dynamic and reversible. By simultaneously monitoring Prp22 and individual spliceosome subcomplexes, I was able to frame these Prp22 binding events in context relative to specific steps in spliceosome assembly and splicing. These experiments provide insight into how Prp22 promotes mRNA release from the spliceosome and maintains splicing fidelity.

Activation of mTORC1 Improves Cone Cell Metabolism and Extends Vision in Retinitis Pigmentosa Mice: A Dissertation

Mon, 08/15/2016 - 11:28am

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.

The Impact of mTORC2 Signaling on the Initiation and Progression of KRAS-Driven Pancreatic Neoplasias: A Dissertation

Mon, 08/15/2016 - 11:28am

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts.

Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR.

In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells.

These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

A Novel SMC-Like Protein Modulates C. Elegans Condensin Functions: A Dissertation

Mon, 08/15/2016 - 11:28am

Chromatin is organized dynamically to accommodate different biological processes. One of the factors required for proper chromatin organization is a group of complexes called condensins. Most eukaryotes have two conserved condensins (I and II) required for chromosome segregation. C. elegans has a third condensin (IDC) that specializes in dosage compensation, a process that down-regulates X gene dosage in XX hermaphrodites to match the dosage in XO males. How the three condensins are regulated is not well understood. Here, I present the discovery and characterization of a novel condensin regulator, SMCL-1.

We identified SMCL-1 through purification of a MAP-tagged condensin subunit. Condensins are comprised of SMC ATPases and regulatory CAP proteins; SMCL-1 interacts most abundantly with condensin SMC subunits and resembles the ATPase domain of SMC proteins. Interestingly, the SMCL-1 protein has residues that differ from SMC consensus and potentially render SMCL-1 incapable of hydrolyzing ATP. Worms harboring smcl-1 deletion are viable and show no detectable phenotype. However, deleting smcl-1 in a condensin hypomorph mildly suppresses condensin I and IDC mutant phenotypes, suggesting that SMCL-1 functions as a negative regulator of condensin I and IDC. Consistent with this, overexpression of SMCL-1 leads to condensin loss-of-function phenotypes such as lethality, segregation defects and disruption of IDC localization on the X chromosomes. Homology searches based on the unique ATPase domain of SMCL-1 reveal that SMCL-1-like proteins are present only in organisms also predicted to have condensin IDC. Taken together, we conclude that SMCL-1 is a negative modulator of condensin functions and we propose a role for SMCL-1 in helping organisms adapt to having a third condensin by maintaining the balance among three condensin complexes.

Conceptualizing, Understanding, and Assessing Research Literacy in a Diverse Population: A Dissertation

Mon, 08/15/2016 - 11:28am

Background: Racial and ethnic minorities are under-represented participants in health-related research. Comprehension and understanding of the research process are a barrier to research participation. A potential approach to engaging underserved populations in research is through improving research literacy, which we define as “the capacity to obtain, process and understand basic information needed to make informed decisions about research participation.”

Methods: Through primary data collection and mixed-methods approaches, this doctoral thesis seeks to: 1) define and conceptualize the domains, determinants, and impacts of research literacy through the development of a multi-component comprehensive framework, 2) operationalize research literacy by developing and psychometrically testing the Research Literacy Scale, and 3) quantify differences in research literacy, measured by the Research Literacy Scale, by race/ethnicity, race-related factors, and other socio-demographic factors.

Results: We created a framework outlining eight domains of research literacy and multi-faceted influences of societal, community, researcher, and participant factors that may influence an individual’s level of research literacy. The Research Literacy Scale created is comprised of 16 items, with a KR-20 estimate of 0.81 and test-retest reliability of 0.84. We found differences in mean scale scores by race/ethnicity, age, education, income, and health literacy (all p < 0.01). African-Americans and Latinos have lower research literacy scores, as compared to non-Latino Whites. Race-consciousness was associated with research literacy score.

Conclusions: This study is the first to define, assess, and quantify factors associated with research literacy in a diverse community sample and may provide insights on approaches to enhance minority engagement in health-related research.

Exploring New Strategies to Overcome Resistance in Glioblastoma Multiforme: A Dissertation

Mon, 08/15/2016 - 11:28am

Glioblastoma multiforme (GBM) tumors are highly malignant in nature and despite an aggressive therapy regimen, long–term survival for glioma patients is uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. This creates the need to investigate new therapies for enhancing GBM treatment outside of the standard of care, which includes Temozolomide (TMZ). Our lab focused on two novel strategies to overcome resistance in GBMs. In our first approach, the cellular responses of GBM cell lines to two new TMZ analogues, DP68 and DP86, are reported. The efficacy of these compounds was independent of DNA repair mediated by Methyl Guanine Methyl Transferase (MGMT) and the mismatch repair (MMR) pathway. DP68 or DP86 treated cells do not give rise to secondary spheres, demonstrating that they are no longer capable of self-renewal. DP68-induced damage includes interstrand DNA crosslinks and exhibits a distinct S-phase accumulation before G2/M arrest; a profile that is not observed for TMZ-treated cells. DP68 induces a strong DNA damage response and suppression of FANCD2 expression or ATR expression/kinase activity enhanced the anti-GBM effects of DP68. Collectively, these data demonstrate that DP68, and to a lesser extent DP86, are potent anti-GBM compounds that circumvent TMZ resistance and inhibit recovery of cultures. Our second approach stems from a previous discovery in our lab which demonstrated that the combination of TMZ with Notch inhibition, using a gamma secretase inhibitor (GSI), enhances GBM therapy. Efficacy of TMZ + GSI treatment is partially due to GBM cells shifting into a permanent senescent state. We sought to identify a miR signature that mimics the effects of TMZ + GSI as an alternative vi approach to enhance GBM therapy. MiR-34a expression was highly upregulated in response to TMZ or TMZ + GSI treatment. Exogenous expression of miR-34a revealed that it functions as a tumor suppressor and mimicked the in vitro effects of TMZ + GSI treatment. Additionaly, miR-34a overexpression leads to the downregulation of Notch family members. Together these two studies contribute to our understanding of the complex mechanisms driving resistance in GBM tumors and suggest strategies to develop more effective therapies.

Frailty and Outcomes in Liver Transplantation: A Dissertation

Mon, 08/15/2016 - 11:27am

In recent years, the transplant community has explored and adopted tools for quantifying clinical insight into illness severity and frailty. This dissertation work explores the interplay between objective and subjective assessments of physical health status and the implications for liver transplant candidate and recipient outcomes. The first aim characterizes national epidemiologic trends and the impact of Centers for Medicare and Medicaid quality improvement policies on likelihood of waitlist removal based on the patient being too frail to benefit from liver transplant (“too sick to transplant”). This aim includes more than a decade (2002–2012) of comprehensive national transplant waitlist data (Scientific Registry of Transplant Recipients (SRTR)). The second aim will assess and define objective parameters of liver transplant patient frailty by measuring decline in lean core muscle mass (“sarcopenia”) using abdominal CT scans collected retrospectively at a single U.S. transplant center between 2006 and 2015. The relationship between these objective sarcopenia measures and subjective functional status assessed using the Karnofsky Functional Performance (KPS) scale are described and quantified. The third aim quantifies the extent to which poor functional status (KPS) pre-transplant is associated with worse post-transplant survival and includes national data on liver transplantations conducted between 2005 and 2014 (SRTR). The results of this dissertation will help providers in the assessment of frailty and subsequent risk of adverse outcomes and has implications for strategic clinical management in anticipation of surgery. This research will also to serve to inform national policy on the design of transplant center performance measures.

Barriers and Facilitators to Deaf Trauma Survivors’ Help-Seeking Behavior: Lessons for Behavioral Clinical Trials Research: A Master’s Thesis

Mon, 08/15/2016 - 11:27am

Deaf individuals experience significant obstacles to participating in behavioral health research when careful consideration is not given to accessibility in the design of study methodology. To inform such considerations, we conducted a secondary analysis of a mixed-methods study that explored 16 Deaf trauma survivors’ help-seeking experiences. Our objective was to identify key findings and qualitative themes from consumers' own words that can be applied to the design of behavioral clinical trials methodology. In many ways, the themes that emerged are what we would expect of any research participant, Deaf or hearing – a need for communication access, empathy, respect, strict confidentiality procedures, trust, and transparency of the research process. However, additional considerations must be made to better recruit, retain, and engage Deaf trauma survivors. We summarize our findings in a “Checklist for Designing Deaf Behavioral Clinical Trials” to operationalize the steps researchers should take to apply Deaf-friendly approaches in their empirical work.

Research Participation Decision-Making Among Youth and Parents of Youth with Chronic Health Conditions: A Dissertation

Mon, 08/15/2016 - 9:05am

The purpose and aims of this qualitative descriptive study were to describe how past experiences with research (including communication, information, values and support) may contribute to research fatigue among youth and parents of youth with HIV, CF, and T1D. Eighteen parents and youth were purposively recruited from outpatient subspecialty clinics at a major academic medical center. They took part in qualitative interviews, completed a demographics form, and the Decisional Conflict Scale. Youth participants also completed the Erikson Psychosocial Stage Inventory. Two major themes emerged: blurred lines and hope for the future. Research fatigue was not found in this sample. Results point to challenges with informed consent in settings where research and clinical care are integrated, and suggest that protective factors allow for continued participation without excess burden on youth and parents. Strategies to minimize research fatigue and support engagement in research are offered.

A Pilot Study of the Pharmacogenetics of Ketamine-Induced Emergence Phenomena: A Dissertation

Mon, 08/15/2016 - 9:05am

Background: Up to 55% of patients administered ketamine, experience an emergence phenomena (EP) that closely mimics schizophrenia and increases their risk of injury. While genetics accounts for about 50% of severe adverse drug reactions, no studies have investigated genetic association of ketamine-induced EP in healthy patients. Ketamine is metabolized by CYP 2B6 enzymes and CYP 2B^8^ allele significantly alter ketamine metabolism. In addition, ketamine exerts most of its effects by inhibiting the N-methyl-D-aspartate receptor (NMADR), and NMDAR genes (GRIN2B) are associated with learning and memory impairment and schizophrenia.

Purpose: To investigate the relationship between CYP2B6*6 and GRIN2B single nucleotide polymorphisms (SNPs) and ketamine-induced emergence phenomena (EP).

Methods: This cross-sectional pharmacogenetic study recruited 75 patients having minor orthopedic, hand, foot, anorectal surgeries from two outpatient surgical centers. EP was measured with the Clinician Administered Dissociative State Scale (CADSS). DNA was genotyped using standard Taqman assays and protocols. Genetic association of CYP2B6*6 and GRIN2B (rs1019385 & rs1806191) SNPs and ketamine induced EP occurrence and severity were tested using multivariate logistic and linear regression, adjusting for age, ketamine dose, duration of anesthesia, and time since ketamine administration.

Results: Forty-seven patients (63%) received ketamine and were genotyped. Nineteen EP cases were identified (CADSS > 4), leaving 28 non-EP controls. For our population, CADSS has an internal consistency reliability Cronbach’s alpha of 0.82, and could reliably distinguish ketamine from non-ketamine cases. Occurrence and severity of EP were not associated with CYP2B6*6 or GRIN2B (p > 0.1). Models removing genotype and containing age, ketamine dose, duration of v anesthesia, and time since ketamine administration significantly predicted EP occurrence (p = 0.001) and severity (p = 0.007). Presence and severity of EP did not affect patient satisfaction with care.

Discussion: Younger age, higher dose and longer duration of anesthesia significantly predicted EP occurrence and severity among our sample. This study provides effect size estimates useful for the design of adequately powered future genetic association studies. The feasibility of recruitment from patients undergoing elective, outpatient surgeries and ease of post-operative EP assessment with CADSS supports our approach. However, the small sample size may have limited about ability to determine significant differences.

Conclusion: Fully powered studies are needed to investigate this important phenomena. Determining factors for anesthesia-related EP symptoms may reduce risks and costs associated with this adverse medication effect.

Gestational Age Estimation Based on Fetal Pelvimetry on Fetal Ultrasound in Iraqi Women

Fri, 08/12/2016 - 9:50am

Ultrasound is an integral part of obstetric practice, and assessment of gestational age (GA) is a central element of obstetric ultrasonography. Sonographic estimation of GA is derived from calculations based on fetal measurements. Numerous equations for GA calculation from fetal biometry have been adopted in routine practice. This study reports a new method of estimating GA in the second and third trimester using interischial distance (IID), the distance between the two ischial primary ossification centers, on fetal ultrasound. Four hundred women with uncomplicated normal singleton pregnancies from 16 weeks to term were examined. Standard fetal obstetric ultrasound was done measuring biparietal diameter (BPD) and femur length (FL) for each fetus. The IID, in millimeters, was correlated with the GA in weeks based upon the BPD and FL individually, and the BPD and FL together. Statistical analysis showed strong correlation between the IID and GA calculated from the FL with correlation coefficient (r =0.989, P < 0.001). Strong linear correlation was also found between the IID and GA based upon BPD and BPD+FL. Further statistical analysis using regression equations also showed that the IID was slightly wider in female fetuses, but this difference was not statistically significant. Resulting from this analysis, we have arrived at an easy-to-use equation: GA Weeks = (IID mm + 8) ±1 week. We feel this method can be especially applicable in the developing world, where midwives may not have access to software for fetal biometry in their basic handheld ultrasound machines. Even more sophisticated machines may not come with loaded software for obstetrics analysis. There are several limitations to this study, discussed below. We recommend further studies correlating the IID with other biometric parameters.

HIV type 1 (HIV-1) proviral reservoirs decay continuously under sustained virologic control in HIV-1-infected children who received early treatment

Fri, 08/05/2016 - 1:56pm

BACKGROUND: Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)-infected infants controls HIV-1 replication and reduces mortality.

METHODS: Plasma viremia (lower limit of detection, < 2 copies/mL), T-cell activation, HIV-1-specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1-infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth.

RESULTS: Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1-specific immune responses.

CONCLUSIONS: Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies.

Computational Modeling of T Cell Receptor Complexes

Fri, 08/05/2016 - 1:56pm

T-cell receptor (TCR) binding to peptide/MHC determines specificity and initiates signaling in antigen-specific cellular immune responses. Structures of TCR-pMHC complexes have provided enormous insight to cellular immune functions, permitted a rational understanding of processes such as pathogen escape, and led to the development of novel approaches for the design of vaccines and other therapeutics. As production, crystallization, and structure determination of TCR-pMHC complexes can be challenging, there is considerable interest in modeling new complexes. Here we describe a rapid approach to TCR-pMHC modeling that takes advantage of structural features conserved in known complexes, such as the restricted TCR binding site and the generally conserved diagonal docking mode. The approach relies on the powerful Rosetta suite and is implemented using the PyRosetta scripting environment. We show how the approach can recapitulate changes in TCR binding angles and other structural details, and highlight areas where careful evaluation of parameters is needed and alternative choices might be made. As TCRs are highly sensitive to subtle structural perturbations, there is room for improvement. Our method nonetheless generates high-quality models that can be foundational for structure-based hypotheses regarding TCR recognition.