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Introduction There is ongoing debate over whether to fix asymptomatic contralateral inguinal hernias during repair of the presenting hernia. This study reviewed the practice of one high-volume hernia surgeon, comparing unilateral and bilateral laparoscopic repairs, to establish if bilateral repair is associated with an increased risk of post-operative complications compared to unilateral repair. Material & Methods All patients operated on within a 5-year period were audited and subcategorised depending on whether they underwent unilateral or bilateral repair. Data was collected on complication rates, rates of recurrence, readmission within 30 days and duration of operation. Results 186 patients underwent repair of 265 hernias. 79 patients underwent bilateral repair. 25 patients suffered a complication (13.4%). Complication rates were 11.2% and 16.5% in the unilateral group and bilateral group respectively (p=0.50). These included superficial infection (1.9% vs 1.3%, p=0.75), haematoma (5.6% vs 7.6%, p=0.59), seroma (2.8 vs 7.6%, p=0.13), and chronic pain (0% vs 2.5%, p=0.01). There were no mesh infections, persisting numbness or explantation in either arm. In both groups, median nights stayed was 0. At median length of follow-up of 1.8 years, recurrence rates were 1.9% vs 0% in the unilateral and bilateral arms respectively (p=0.22). Conclusions There was no significant difference in rates of complications between the bilateral and unilateral arms. Opportunistic repair may reduce risk of future surgeries and morbidity and does not appear to be associated with increased complications. Surgeons may wish to consider this when consenting patients.

Introduction This study aims to understand whether previous abdomino-pelvic (AP) surgery is a predictor of peri-operative complication in inguinal hernia surgery and if there are discrepancies between laparoscopic or open repairs. Material & Methods All patients operated on within a 5-year period were audited and subcategorised based upon history of AP surgery. Patients were also categorised into laparoscopic and open groups. Data collected included complication rates, recurrence rates and readmission within 30 days. Results 205 patients underwent repair of 288 hernias. 80 (39%) patients had a history of AP surgery. 32 patients suffered a complication (15.6%). 85% of the repairs were laparoscopic in the AP group vs 96.4% in the non-AP group (p=0.02). Complication rates were 17.5% and 14.4% in the AP group and non-AP group respectively (p=0.55). These included superficial infection (0% vs 2.4%, p=0.16), haematoma (11.3% vs 4.8%, p=0.08), seroma (5.0% vs 4.8%, p=0.95), persisting numbness (0% vs 0.8%, p=0.42), and chronic pain (1.3% vs 0.8%, p=0.75). There were no mesh infections or explantation in either arm. A greater proportion of patients in AP group required overnight stays (32.5% vs 21%, p=0.08). At median follow up 1.8 years, recurrence rates were 3.8% vs 1.6%, p=0.33 in AP and non-AP groups respectively. Conclusions Laparoscopic inguinal hernia is feasible in most patients with a history of AP surgery. Compared to open repair, there were no significant differences in recurrence or complication rates in patients with previous AP surgery. Therefore, previous AP surgery should not be a contraindication for laparoscopic repair.

Introduction This study reviews the practice of a high-volume surgeon, comparing laparoscopic and open surgery for inguinal hernia. In suitable patients the default was laparoscopic Totally Extra Peritoneal (TEP) approach. Materials & Methods Data from patients referred over 5 years with inguinal hernias was collated prospectively and analysed retrospectively. Short and long-term outcomes of laparoscopic and open repairs were compared. Results 205 patients underwent repair of 288 hernias (90.7% laparoscopic, 9.3% open). 83 patients underwent bilateral repair (95.2% laparoscopic). 22 patients underwent repair of 27 recurrent hernias (68% laparoscopic). 1 case (0.5%), started laparoscopically was converted to open. Complication rates were 13.4% vs 36.8% in the laparoscopic and open arms respectively (p Mean stay was significantly longer in open group (p=0.03), median stay was 0 nights in both groups. At median follow up 1.8 years (Interquartile-range 0.9–3.6 years) recurrence rate was 1.1% (0 female, 2 male) and 15.8% (1 female, 2 male) in the laparoscopic and open arms respectively (p Conclusions This series demonstrates the vast majority of inguinal hernias, both primary and recurrent can be treated with laparoscopic TEP repair. Higher recurrence and complication rates were associated with open repair, with a caveat of strong selection bias.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Introduction Apert syndrome is an autosomal dominant malformation syndrome, accounting for 4.5% of all craniosynostoses. Raised intracranial pressure (ICP) in Apert syndrome has a multifactorial aetiology, with an incidence of up to 45% if left untreated [1, 2]. Raised ICP can be determined clinically, with non-invasive and invasive methods. In this study, we want to assess whether the use of CT scans is reliable in identifying changes in ICP. Method Pre and postoperative CT scans for 13 Apert syndrome patients who had posterior vault expansion were assessed and graded for severity of intracranial pressure (ICP). The grading system used was departmental specific and the assessment was carried out by a single clinician on different brain structures. This process was repeated on the same patients, using the same CT scans, 4 months later to determine consistency and repeatability. The relationship between the pre and postoperative scans was explored using the chi squared test. Intra-observer variability was assessed using Kappa statistics [SS1]. Results There was no statistically significant difference between the pre and postoperative CT scan grading. Across instances, only one assessed structure had a p-value Conclusions Assessment of CT scans is not a reliable method to determine changes in intracranial pressure in Apert syndrome patients who have had a posterior vault expansion.

The Tracking and Evaluation Core of Rhode Island Advance-CTR conducted an online needs assessment survey at the program’s inception in 2016 and again in 2021. Now dealing with well-established support systems provided by the grant, we were particularly interested in how the perceived needs of the research community in Rhode Island might have changed over five years. Specifically, what barriers have been reduced or eliminated and which have persisted or increased? How do those barriers vary by demographic status and what implications do those differences have for the CTR? An online survey was completed by 199 researchers, who reported the extent to which they perceived the lack of access to a range of research supports as a barrier to conducting research at their institution. Overall, researchers indicated statistically significant changes from 2016 to 2021 such that a lack of pilot project funding and proposal development support had decreased as barriers, while space for research, and advice on commercial development, had increased. Statistically significant differences in the salience of particular barriers by some demographic variables were also noted and the results of this study suggest Centers for Clinical and Translational Research can have salutary effects on the research paradigm within their partnering institutions in a relatively short time.

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies.

Background Intramuscular injections (IMI) are used to deliver preventive and curative treatment in sexual health clinics, but can result in severe and prolonged pain for patients, cause significant anxiety, and limit future treatment options. Methods A survey to assess the importance of interventions to reduce injection pain amongst sexual health clinic attendees, and an integrative literature review exploring IMI delivery. Results Two hundred and three patients who were receiving an IMI participated in the survey. More than half (27/52[52%]) of patients receiving gluteal injections and a third (49/151[32%]) of those receiving deltoid injections reported that approaches to reduce IMI pain would be ‘important’ or ‘very important’. A variety of IMI techniques with the potential to reduce injection pain were identified in the review. The Z track injection technique (stretching the overlying skin and subcutaneous tissues to one side just prior to injection) was widely recommended, but with limited supporting evidence, and adopted only to a small extent in clinical practice. More evidence was available supporting the use of manual pressure over the injection site or Helfer (rhythmic) tapping on the injection site before injection but this was mostly from small studies. Other factors including patient demographics, anthropometry, injectate volume, needle length, and muscle site affected perceived injection pain and risk of subsequent complications. Conclusion Intramuscular injection pain is of importance to sexual health patients. The use of localised pressure over the injection site prior to injection may reduce pain but high quality studies are required to confirm the optimal injection technique.

Background Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations. Results We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities. Conclusions The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa.

In evaluation research, "programs" are often conceptualized as clearly bounded, narrow in scope, focused on specific outcomes, using a well-defined linear causal model, and hence, suitable for standard evaluation methods. The evaluation work reported here was carried out in a more challenging context, where large, complex, interwoven systems were targets for change as a means to influence a diffuse array of outcomes. Our evaluation of an NIH-funded program to improve statewide infrastructure for clinical and translational health research ("Advance-CTR") used qualitative data provided by investigators who used the program's services, were funded awardees, or were members of an internal advisory committee (leadership representatives from partnering institutions). We examined perceived barriers to systemic changes to enhance research, as well as how systems have changed due to the Rhode Island Advance-CTR program's efforts, to what degree, and with what effects. Using the causal logic of our program to connect these more distal systemic outcomes to the services and components of Advance-CTR, we discuss the effects this program has had on researchers and their environments, contributing to the development of sustainable programs of research that ultimately improve the health and well-being of our state's residents.

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

Case Summary: A 23-year-old female with common variable immunodeficiency (CVID), presented with intermittent epigastric pain, which would eventually spread to her entire abdomen. She had night sweats, chills, and regular stools. She had been evaluated twice over the last two months for posterior cervical lymphadenopathy and similar abdominal symptoms, which were attributed to a possible EBV infection. Her exam was notable for diffuse abdominal tenderness, worse in the epigastric region, without rebound or guarding. Additionally, she had a non-tender fixed lymph node in the right posterior cervical chain. Her medical …

BackgroundWith the emergence of the Coronavirus vaccine and the clear and important role it provides in maintaining the health of individuals and the population (1), it is important that our patients receive or are recommended the vaccine. We here document a case series of patients who have long-term quiescent rheumatoid arthritis who then experienced a flare of disease activity after receiving a vaccine. This information is important to understand as it allows informed discussions regarding the side effect profile of the vaccine and also the influence this may have on the patient’s disease control and future management options.ObjectivesTo understand the changes to control of rheumatoid arthritis in those experiencing a flare of disease after receiving the coronavirus vaccine.MethodsPatients were reviewed in clinic as part of standard care. Individuals with Rheumatoid Arthritis who had experienced a flare defined as a self-reported disease activity score of >5.1 in otherwise stable disease were noted and their response to treatment reviewed. This was a review of usual clinical practice and did not alter the treatment undertaken or monitoring of the patent. Information obtained was through the consultation with a rheumatologist and the data was collected retrospectively through review of clinical notes and clinic letters.ResultsThe table below outlines the patient details and treatment responses.All flares occurred within two weeks of receiving the vaccine. Of the patients who flared, two required short term steroid treatment; three an increase in the usual medications and one who had been in disease remission to re-start previous therapy. More patients in the Pfizer vaccine group required an escalation of usual care compared to the AstraZeneca vaccine group.Table 1Antibody statusVaccine brandTime since diagnosisTime since last flareFlare after 1st or 2nd doseUsual treatmentFlare managementSeropositive, anti-CCP positiveAstraZeneca13 years3 years1stEtanercept, MethotrexateIntramuscular MethylprednisoloneSeropositive, anti-CCP positiveAstraZeneca11 years4 years1stEtanercept, SulfasalazineIntramuscular Methylprednisolone, local joint injectionSeronegative, anti-CCP negativeAstraZenica13 years2 years1stMethotrexateIncrease methotrexate doseSeronegative, anti-CCP positivePfizer23 years11 years1stInfliximabLocal joint injection, reduce interval of infusionsSeronegative, anti-CCP positivePfizer7 years3 years2ndSulfasalazine, hydroxychloroquineIncrease Sulfasalazine doseSeropositive, anti-CCP positivePfizer7 years4 years2ndnilRe-start methotrexate, hydroxychloroquineConclusionWe report 6 cases of rheumatoid arthritis flare soon after receipt of the coronavirus vaccine. In all, disease control was returned with minimal changes to treatment, 42% of those requiring either no treatment or an intramuscular steroid injection alone. Therefore, we recommend that clinicians should counsel patients of this potential effect, but continue to advocate the vaccine, as the risk of complications to their underlying arthritis is very low and seemingly easily treatable.References[1]Tenforde MW, Olson SM, Self WH, Talbot HK, Lindsell CJ, Steingrub JS, et al. Effectiveness of Pfizer-BioNTech and Moderna Vaccines Against COVID-19 Among Hospitalized Adults Aged ≥65 Years - United States, January-March 2021. MMWR. Morbidity and mortality weekly report. 2021; 70 (18): 674-679. Available from: doi: 10.15585/mmwr.mm7018e1Disclosure of InterestsNone declared

BackgroundAlthough it may be difficult to detect changes in spinal mobility on the short term, spinal mobility is considered an important measure to assess the efficacy of drugs used to treat axial spondyloarthritis (axSpA). However, few studies evaluated the long-term impact of biologic treatment on spinal mobility.ObjectivesTo describe the long-term effect of TNF inhibitors (TNFi) on spinal mobility in patients with axSpA, and to determine whether the use of TNFi treatment influences spinal mobility, and if this due to a direct or indirect effect (mediated by disease activity).MethodsWe performed a retrospective observational study, using data collected from patients with a clinical diagnosis of axSpA treated with TNFi at a tertiary care centre where disease activity and metrology assessments are routinely done. Adult patients with at least two Bath Ankylosing Spondylitis Metrology Index (BASMI) measurements were included. Disease activity was measured using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score C-reactive protein (ASDAS). The longitudinal association between TNFi and improvement in BASDAI/ASDAS was tested using a linear mixed effects model with BASMI as dependent variable. To test whether TNFi had a direct effect on BASMI, not mediated by disease activity, we tested that TNFi treatment was not conditionally independent of BASMI given BASDAI/ASDAS (Figure 1). We tested whether the nodes TNFi and BASMI were disconnected if we removed BASDAI and ASDAS. To test this conditional independence, we first built a linear mixed effects model for BASMI given BASDAI or ASDAS when the patient was under TNFi and used this model to predict a 95% confidence interval (CI) for BASMI given the data for BASDAI/ASDAS when the patient was without TNFi. We checked whether the true value of BASMI lay within this 95% CI and performed a hypothesis test for binomial distribution where H0: p=0.95. To test for the indirect effect of TNFi on BASMI reduction, mediated through the disease activity, we regressed BASMI on BASDAI/ASDAS, TNFi (if there was a direct effect), demographics, presence of radiographic (r-) axSpA and HLA-B27 positivity, using a linear mixed effects model adjusted for within-patient correlation.Figure 1.Indirect effect of TNFi on BASMI (represented by the full line), through the influence of TNFi on disease activity, adjusted by other confounders and direct effect of TNFi on BASMI (dashed line), independently of disease activity.ResultsData from 188 patients and 1326 visits were analysed. Mean age was 45.6 (SD 11.6) years, mean disease duration was 15.8 (SD 9.64) years, 152 (80.9%) were male, 120 (73.6%) had r-axSpA, and 83 (74.8%) were HLA-B27 positive. Mean follow-up time was 8.0 (SD 4.4) years, ranging from 0.8 to 18.2 years. Treatment with TNFi was significantly associated with long-term improvement in BASMI (B=-0.423, 95% CI=[-0.553,-0.292], pConclusionTNFi are effective at improving BASMI in patients with axSpA, in a real-life setting. This effect is mainly explained by the reduction in disease activity. However, a direct effect of TNFi on BASMI could also be demonstrated, when disease activity was measured by BASDAI, suggesting that ASDAS captures additional factors that can influence spinal mobility. These potential factors deserve further investigation, but they could for example include biomechanical properties of tendons and myofascial tissue.Disclosure of InterestsAna Sofia Pinto: None declared, Bohao Yao: None declared, Claire Harris: None declared, Rhys Hayward: None declared, Andrew Keat: None declared, Pedro Machado Speakers bureau: Received consulting/speaker’s fees from Abbvie, BMS, Celgene, Eli Lilly, Galapagos, Janssen, MSD, Novartis, Orphazyme, Pfizer, Roche and UCB, all unrelated to thismanuscript, Consultant of: Received consulting/speaker’s fees from Abbvie, BMS, Celgene, Eli Lilly, Galapagos, Janssen, MSD, Novartis, Orphazyme, Pfizer, Roche and UCB, all unrelated to thismanuscript

Introduction: Advance Clinical and Translational Research (Advance-CTR) serves as a central hub to support and educate clinical and translational researchers in Rhode Island. Understanding barriers to clinical research in the state is the key to setting project aims and priorities. Methods: We implemented a Group Concept Mapping exercise to characterize the views of researchers and administrators regarding how to increase the quality and quantity of clinical and translational research in their settings. Participants generated ideas in response to this prompt and rated each unique idea in terms of how important it was and feasible it seemed to them. Results: Participants generated 78 unique ideas, from which 9 key themes emerged (e.g., Building connections between researchers). Items rated highest in perceived importance and feasibility included providing seed grants for pilot projects, connecting researchers with common interests and networking opportunities. Implications of results are discussed. Conclusions: The Group Concept Mapping exercise enabled our project leadership to better understand stakeholder-perceived priorities and to act on ideas and aims most relevant to researchers in the state. This method is well suited to translational research enterprises beyond Rhode Island when a participatory evaluation stance is desired.

Background We compared vaginal microbial communities in postmenopausal black and white women. Methods Shotgun sequencing of vaginal swabs from postmenopausal women self-identified as black or white was compared using MiRKAT. Results Vaginal community dominance by Lactobacillus crispatus or Lactobacillusgasseri was more common in 44 postmenopausal black women (n = 12, 27%) than among 44 matched white women (n = 2, 5%; P = .01). No individual taxa were significantly more abundant in either group. Conclusions We identified small overall differences in vaginal microbial communities of black and white postmenopausal women. L. crispatus dominance was more common in black women. Clinical Trials Registration NCT02516202 (MsFLASH05) and NCT01418209 (MsFLASH03).

Importance. Observational studies have suggested that the use of radial artery grafts for coronary artery bypass grafting may improve clinical outcomes compared with the use of saphenous vein grafts, but this has not been confirmed in randomized trials.Objective. To compare clinical outcomes between patients receiving radial artery or saphenous vein grafts for coronary artery bypass grafting at long-term follow-up.Design, Setting, and Participants. Patient-level pooled analysis comparing radial artery and saphenous vein grafts in adult patients undergoing isolated coronary artery bypass grafting from five countries (Australia, Italy, Serbia, South Korea, and United Kingdom; enrollment years: 1997 to 2009, follow-up completed in 2019).Intervention. Patients were randomized to receive either radial artery or saphenous vein grafts for coronary artery bypass grafting.Main outcomes and measures. The primary outcome was a composite of death, myocardial infarction or repeat revascularization, and the secondary outcome was a composite of death or myocardial infarction.Results. 1036 patients (534 radial artery group) were randomized (mean age 66.6 vs 67.1 years, males 70.4% vs 69.9% in the radial artery and the saphenous vein groups, respectively); 942 (90.9%) of the originally randomized patients completed 10 years of follow-up (510 radial artery group). At a median follow-up of 10 years (interquartile range 10-11), the use of the radial artery was associated with a statistically significant reduction in the incidence of the composite outcome of death, myocardial infarction or repeat revascularization (220 vs 237 events; 41 vs 47 events per 1000 patient-years; hazard ratio[HR] 0.73, 95%confidence interval[CI] 0.61-0.88, pConclusions and relevance. In this individual participant data meta-analysis with a median follow up of 10 years, among patients undergoing coronary artery bypass grafting, the use of the radial artery compared with saphenous vein grafts was associated with a lower risk of a composite of cardiovascular outcomes.

Lactobacillus iners is a common member of the human vaginal microbiota, with a genome size smaller than that of other lactobacilli. Here, we report the complete genome sequences of six L. iners strains isolated from different vaginal swab specimens. Three strains were found to harbor ∼100-kbp plasmids, which were not known previously.

Plants possess a complex network of mechanisms to utilize and, if necessary, detoxify metals. Plants utilize constitutive basal tolerance mechanisms to maintain appropriate internal metal levels under normal conditions. However, adaptive hyper-tolerance mechanisms are used in order to tolerate excess metal exposure. The production of metal binding chelators could be one way to convey these tolerances. Chelator production of field and greenhouse-derived materials was investigated to determine any multi-metal hyper-tolerances in different populations of the grass Deschampsia cespitosa (L.) Beauv. Plant tissue was collected from metal-contaminated mine sites, and from specimens grown in metal exposure hydroponic experiments. The chelator metabolites from these samples were simultaneously analyzed using HPLC-tandem mass spectrometry. In the hydroponic grown grass, histidine was produced at high concentrations solely in the hyper-tolerant populations during metal exposure. In all of the populations, the responses of chelators were metal-specific, where levels of nicotianamine were at high concentrations during Ni exposure, and levels of phytochelatins were high during Cu exposure. Moreover, a similar pattern of chelator production was seen in the root specimens collected from mine sites contaminated with Ni and (or) Cu. Histidine was the strongest Ni chelator involved in adaptive hyper-tolerance, while constitutive basal tolerance to Ni and Cu was observed via the responses of nicotianamine and phytochelatin, respectively.

A change in cardiac surgery practice over the past decade has seen an increase in urgent or inpatient referrals for surgery, with antiplatelet therapy often continued up until surgery. This study aims to identify the optimal timing for administration of aspirin to minimise risk of perioperative morbidity and mortality.From a prospectively compiled database collected by the Australian and New Zealand Society of Cardiac and Thoracic Surgeons, we identified 8294 patients undertaking combined CABG and valve or isolated valve procedures while discontinuing aspirin. Time points for cessation of antiplatelet therapy were categorised as follows:2 days, 3-7 days or7 days preoperatively. We evaluated the association of adverse in-hospital events and intermediate term survival in each time category.Discontinuing aspirin 3 to 7 days from surgery decreased rates of perioperative MI (HR=0.300, p=0.027), return to theatre (HR=0.560, p=0.002) reduced drain output (HR=0.757, p=0.000) and red blood cell and platelet transfusions (HR=0.719, p=0.000 and HR=0.604, p=0.000 respectively) compared to patients continuing aspirin until2 days from the procedure. Stopping aspirin2 days from the date of surgery increased risk of perioperative MI (HR=5.919, p=0.000), reoperation for bleeding (HR=2.076, p=0.001), returning to theatre (HR=1.781, p=0.000), ICC drain losses (HR=1.337, p=0.000) and transfusion demands for red blood cells (HR=1.381, p=0.000) and platelets (HR=1.450, p=0.000) when compared to those discontinuing aspirin7 days from surgery.Late discontinuation of aspirin before combined coronary artery bypass graft and valve procedures results in greater rates of bleeding and transfusion requirements. Earlier discontinuation of aspirin results in no benefit in intermediate term survival.

IMPACT: This work has begun to provide the foundation for better ensuring that translational research funded and supported by our IDeA-CTR grant is more directly addressing community- and stakeholder-authored health priorities. OBJECTIVES/GOALS: In order to effectively engage diverse, societal perspectives, we aimed to determine the relevance and feasibility of purposefully aligning translational research with health priorities adopted by the RI Department of Health, health-focused organizations, and community leaders. METHODS/STUDY POPULATION: Individuals from 27 community organizations in RI were asked, ‘What are your health related goals for your community’ and submitted responses online for 2 weeks. Participants generated 71 goals which they sorted into meaningful clusters and rated for importance and feasibility. Clusters were contrasted with RI health priorities to gauge alignment and saturation. In the next phase of this project, researchers and service users funded by Advance-CTR will be asked in routinely administered surveys how their current work may align with RI health goals and whether their future work can feasibly be connected to those priorities. RESULTS/ANTICIPATED RESULTS: Using Group Concept Mapping software, the 71 health goals identified by community organization representatives were fit into an 8-cluster model. Results suggested highest importance placed on Accessible & Healthy Housing (M=4.12, SD=0.29), Community (M=4.08, SD=0.28), Youth (M=4.04, SD=0.49) and Mental Health (M=4.03, SD=0.46). State agency priorities were found to overlap substantially with clusters defined by community leaders. We expect researchers will rate clusters differently, and find some community-endorsed health goals more relevant to their work than others. Perceived feasibility of tailoring future research to state health goals is expected to vary widely by item and researcher. DISCUSSION/SIGNIFICANCE OF FINDINGS: We intend to: 1) facilitate discussions about successes and challenges of translating community-authored priorities into research, and 2) foster better understanding between researchers and the communities they aim to serve on the role of CTR for addressing health challenges in the state.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

Objectives To compare the physiology of apneic oxygenation with low-tidal-volume (VT) ventilation during harvesting of the left internal mammary artery. Design Prospective, single-center, randomized trial. Setting Single-center teaching hospital. Participants The study comprised 24 patients who underwent elective coronary artery bypass grafting surgery. Interventions Apneic oxygenation (apneic group: 12 participants) and low-VT ventilation (low-VT group: 12 participants) (2.5 mL/kg ideal body weight) for 15 minutes during harvesting of the left internal mammary artery. Measurement and Main Results The primary endpoint was an absolute change in partial pressure of arterial carbon dioxide (PaCO2). Secondary endpoints were changes in arterial pH, pulmonary artery pressures (PAP), cardiac index, and pulmonary artery acceleration time and ease of surgical access. The mean (standard deviation) absolute increase in PaCO2 was 31.8 mmHg (7.6) in the apneic group and 17.6 mmHg (8.2) in the low-VT group (baseline-adjusted difference 14.2 mmHg [95% confidence interval 21.0-7.3], p

International audience; Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

Background Cervicovaginal microbiota domination by Lactobacillus crispatus is associated with beneficial health outcomes, whereas L. iners dominance has more adverse associations. However bacterial vaginosis (BV) treatment with metronidazole (MTZ) typically leads to domination by L. iners rather than L. crispatus. L. iners differs from other lactobacilli by its inability to grow in MRS media. We hypothesized that exploring this growth difference would identify targets for selective L. iners inhibition. Methods Bacteria were grown anaerobically. Nutrient uptake and metabolism were assessed using UPLC-MS/MS and isotopically labeled substrates. Bacterial genome annotation employed Prodigal, Roary, and EggNOG. Competition experiments with mock mixed communities were analyzed by 16S rRNA gene sequencing. We confirmed result generalizability using a diverse collection of South African and North American strains and genomes. Results Supplementing MRS broth with L-cysteine (Cys) or L-cystine permitted robust L. iners growth, while L. crispatus grew without Cys supplementation. Despite their different growth requirements, neither species could synthesize Cys via canonical pathways. Adding the cystine uptake inhibitors S-methyl-L-cysteine (SMC, Fig 1) or seleno-DL-cystine (SDLC) blocked growth of L. iners but not other lactobacilli, suggesting L. iners lacks mechanisms other lactobacilli use to exploit complex exogenous Cys sources. Notably, cydABCD, an operon with Cys/glutathione transport and redox homeostasis activities, is absent from L. iners but present in non-iners Lactobacillus species. Consistent with possible roles for cydABCD in explaining the observed phenotypes, (1) L. iners failed to take up exogenous glutathione and (2) supplementing MRS with reducing agents permitted L. iners growth, which could be blocked by SMC or SDLC. In growth competitions testing L. iners and L. crispatus within mock BV-like communities, SMC plus MTZ outperformed MTZ alone in promoting L. crispatus dominance (Figs 2&3). Figure 1: S-methyl-L-cysteine (SMC) selectively blocks growth of L. iners but not other cervicovaginal Lactobacillus species in cysteine-supplemented MRS broth. Growth was measured by optical density and inhibition calculated relative to Cys-supplemented no-inhibitor control during exponential growth. Values displayed are median (+/- maximum/minimum) for 3 replicates from a single experiment. In all panels, representative data are shown from 1 of >=2 independent experiments for each bacterial strain and media condition. Results are representative of multiple strains for L. iners (n = 16), L. crispatus (n = 7), and L. jensenii (n = 2). Figure 2: Relative abundance of L. crispatus, L. iners, or various BV-associated bacteria in mock bacterial communities grown in rich, non-selective media with or without metronidazole (MTZ) and/or SMC. Relative abundance was determined by bacterial 16S rRNA gene sequencing. Data are shown for three representative mock communities with 5 replicates per media condition. Figure 3: Ratio of L. crispatus to other species in the mock bacterial communities depicted in Figure 2. Statistical significance determined via 1-way ANOVA of log10-transformed ratios with post-hoc Tukey test; selected pairwise comparisons are shown (***, p < 0.001). Conclusion L. iners has unique requirements for exogenous cysteine/cystine or a reduced environment for growth. Targeting cystine uptake to inhibit L. iners is a potential strategy for shifting cervicovaginal microbiota towards L. crispatus-dominant communities. Disclosures Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

It is generally accepted that radial artery (RA) grafts have better mid-term patency rate compared to saphenous vein grafts. However, the clinical correlates of the improved patency rate are still debated. Observational studies have suggested increased survival and event-free survival for patients who receive an RA rather than a saphenous vein, but they are open to bias and confounders. The only evidence based on randomized data is a pooled meta-analysis of 6 randomized controlled trial comparing the RA and the saphenous vein published by the RADial artery International Alliance (RADIAL). In the RADIAL database, improved freedom from follow-up cardiac events (death, myocardial infarction and repeat revascularization) was found at 5-year follow-up in the RA arm. The most important limitation of the RADIAL analysis is that most of the included trials had an angiographic follow-up in the first 5 years and it is unclear whether the rate of repeat revascularization (the main driver of the composite outcome) was clinically indicated due to per-protocol angiographies. Here, we present the protocol for the long-term analysis of the RADIAL database. By extending the follow-up beyond the 5th postoperative year (all trials except 1 did not have angiographic follow-up beyond 5 years), we aim to provide data on the role of RA in coronary artery bypass surgery with respect to long-term outcomes.

Background We used a large patient-level data set including 6 angiographic randomized controlled trials (RCTs) on coronary artery bypass conduits to explore incidence and determinants of coronary graft failure. Methods Patient-level angiographic data of 6 RCTs comparing long-term outcomes of the radial artery and other conduits were joined. Primary outcome was graft occlusion at maximum follow-up. The analysis was divided as (1) left anterior descending coronary (LAD) distribution and (2) non-LAD distribution (circumflex and right coronary artery). Mixed-model multivariable Cox regression including all baseline characteristics with stratification by individual trials was used to identify predictors of graft occlusion. Results Included were 1091 patients and 2281 grafts, consisting of 921 left internal mammary arteries, 74 right internal mammary arteries, 710 radial arteries, and 576 saphenous veins. All left internal mammary arteries were used on the LAD, the other conduits were used on the non-LAD distribution. Mean angiographic follow up was 65 ± 29 months. Occlusion rates were 2.3% for the left internal mammary arteries, 13.5% for the left internal mammary arteries, 9.4% for the right internal mammary arteries, and 17.5% for the saphenous veins. At multivariable analysis, type of conduit used, age, female sex, left ventricular ejection fraction of less than 0.50, and use of the Y graft were significantly associated with graft occlusion in the non-LAD distribution. Conclusions Our analyses showed that failure of the left internal mammary arteries-to-LAD bypass is a very uncommon event. For the non-LAD distribution, the nonuse of radial artery, age, female sex, left ventricular ejection fraction of less than 0.50, and use of the Y graft configuration were significantly associated with midterm graft failure.

Phosphoinositide 3-kinase (PI3K) plays an integral role in lymphocyte function. Mutations in PIK3CD and PIK3R1, encoding the PI3K p110δ and p85α subunits, respectively, cause increased PI3K activity and result in immunodeficiency with immune dysregulation. We describe here the first cases of disseminated and congenital toxoplasmosis in a mother and child who share a pathogenic mutation in PIK3R1 and review the mechanisms underlying susceptibility to severe Toxoplasma gondii infection in activated PI3Kδ syndrome (APDS) and in other forms of primary immunodeficiency.

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human VH-genes. We find that, of six commonly deployed VH sequences, only those CDRs encoded by IGHV1-2∗02 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the VH-gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target., Graphical Abstract, Highlights • Transgenic mice alter VH-gene contribution to human-like BCR repertoires • Specific human VH-genes are needed to engage bloodborne bacterial LPS in vivo • Germline-endowed antigen recognition targets a universally conserved LPS moiety, The ligand binding surface of antibodies is generated by diverse gene-encoded and hypervariable loops. Sangesland et al. demonstrate that certain gene-encoded loops are needed for human antibodies to accommodate the shapes of some bacterial antigens and naturally tune for recognition of conserved microbial features therein.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of Tcell receptor excision circles (TRECs) and Tcell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent Tcell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cellcounts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of Tcell lymphopenia at birth.

OBJECTIVES/SPECIFIC AIMS: We sought to solicit and synthesize stakeholders’ ideas for how the Advance-CTR program can best increase the quality and quality of clinical and translational research in Rhode Island, and to apply these findings to address barriers and strengthen research capabilities across our partner institutions. METHODS/STUDY POPULATION: We utilized a Group Concept Mapping approach, involving university and Institution-based researchers and administrators. The process was conducted using the web-based concept mapping application CS Global Max (Concept Systems, Inc). Respondents were asked to provide their best ideas for promoting clinical and translational research in RI. These ideas were then organized by our project team into a set of unique items for consideration by attendees of an Advance-CTR retreat. Participants were tasked with sorting these ideas by theme (cluster), and were also asked to rate each idea according its importance and feasibility. Using the online software, these clusters and ratings were analyzed to identify key themes and to explore differences among sub-groups. RESULTS/ANTICIPATED RESULTS: The Group Concept Mapping exercise yielded 150 statements that were edited down to 78 unique ideas, and clustered into nine themes (e.g., institutional collaboration, training). Fifty-seven retreat participants completed the sorting and rating tasks of the concept mapping exercise. Overall, ideas rated as highly important and highly feasible included “providing seed grants to encourage new collaborations across basic science,” and “connecting researchers with common interests.” Top rated items varied across institutions and according to respondent demographics, allowing us to consider the unique issues relevant to particular groups. Relative rankings of clusters across groups revealed notable differences, such as higher importance placed on community engagement among administrators as compared with researchers, and differences in needs for internal support for research between universities. DISCUSSION/SIGNIFICANCE OF IMPACT: Group Concept Mapping was an effective and insightful participatory approach to engage our program’s stakeholders in developing ideas and identifying challenges to enhancing clinical and translational research in Rhode Island. Our results have implications for project decision-making and initiatives to facilitate translational research in RI. Thus, results have been presented to the Advance-CTR community via webinar, as well as Advance-CTR project leadership and advisory committees.

Severe combined immunodeficiency (SCID), a primary immunodeficiency arising from variable defects in lymphocyte development and survival, is characterized by significant deficiency of thymus derived (T-) lymphocytes and variable defects in the B-lymphocyte population. Newborn screening for SCID is based on detection of low numbers of T-cell receptor excision circles (TRECs) by real time quantitative PCR (RT-qPCR). This screening allows for early identification of individuals with SCID and other disorders characterized by T-lymphopenia. Higher rates of abnormal screens are commonly seen in premature and critically ill neonates, often representing false positives. It is possible that many abnormal screens seen in these populations are result of conditions that are characterized by systemic inflammation or stress, possibly in the context of stress-induced thymic involution. We present a case of a male infant delivered at 27 weeks, 6 days of gestation, with severe intrauterine growth restriction who had an abnormal TREC screen and amassive perivillous fibrin deposition(MPFD) of the placenta. This association has not been reported previously. We are raising the awareness to the fact that conditions, such as MPFD, that can create adverse intrauterine environment are capable of causing severe stress-induced thymic involution of the fetus which can present with abnormal TREC results on newborn screening.

Background Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs. Results We present a dog gut microbiome gene catalog containing 1,247,405 genes (based on 129 metagenomes and a total of 1.9 terabasepairs of sequencing data). Based on this catalog and taxonomic abundance profiling, we show that the dog microbiome is closer to the human microbiome than the microbiome of either pigs or mice. To investigate this similarity in terms of response to dietary changes, we report on a randomized intervention with two diets (high-protein/low-carbohydrate vs. lower protein/higher carbohydrate). We show that diet has a large and reproducible effect on the dog microbiome, independent of breed or sex. Moreover, the responses were in agreement with those observed in previous human studies. Conclusions We conclude that findings in dogs may be predictive of human microbiome results. In particular, a novel finding is that overweight or obese dogs experience larger compositional shifts than lean dogs in response to a high-protein diet. Electronic supplementary material The online version of this article (10.1186/s40168-018-0450-3) contains supplementary material, which is available to authorized users.