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Family members carrying the same SCN1A variant often exhibit differences in the clinical severity of epilepsy. This variable expressivity suggests that other factors aside from the primary sodium channel variant influence the clinical manifestation. However, identifying such factors has proven challenging in humans.We perform whole exome sequencing (WES) in a large family in which an SCN1A variant (p.K1372E) is segregating that is associated with a broad spectrum of phenotypes ranging from lack of epilepsy, to febrile seizures and absence seizures, to Dravet syndrome. We assessed the hypothesis that the severity of the SCN1A-related phenotype was affected by alternate alleles at a modifier locus (or loci).One of our top candidates identified by WES was a second variant in the SCN1A gene (p.L375S) that was shared exclusively by unaffected carriers of the K1372E allele. To test the hypothesized that L375S variant nullifies the loss-of-function effect of K1372E, we transiently expressed Nav1.1 carrying the two variants in HEK293T cells and compared their biophysical properties with the wild-type (WT) variant, and then co-expressed WT with K1372E or L375S with K1372E in equal quantity and tested the functional consequence. The data demonstrated that co-expression of the L375S and K1372E alleles reversed the loss-of-function property brought by the K1372E variant, whereas WT-K1372E co-expression remained partial loss-of-function.These results support the hypothesis that L375S counteracts the loss-of-function effect of K1372E such that individuals carrying both alleles in trans do not present epilepsy-related symptoms. We demonstrate that monogenic epilepsies with wide expressivity can be modified by additional variants in the disease gene, providing a novel framework for the gene-phenotype relationship in genetic epilepsies.

The increased sensitivity of advanced molecular techniques greatly exceeds the sensitivities of traditional detection methods for infectious agents. This sensitivity causes difficulty in interpreting the biological significance of such detections in fish (and shellfish), especially when the agent(s) cannot be cultured in the laboratory. In the Pacific Northwest, including Canada and Alaska, molecular detections of "new" (unknown or known but discovered in a different geographic location or fish host) potentially infectious agents in fish have received extensive media attention and misinterpretation that call for resource agencies to change current fish health surveillance practices or policies to include these agents. Fish health specialists from several of these agencies and organizations (see Acknowledgments) advise that any policy changes should be made only after further investigations to avoid wasting resources to conduct surveillance for organisms that are not significant to fish health or for noninfectious genetic material that does not represent a viable agent. Molecular detection is not proof of agent viability within or on host tissues and requires further investigation regarding the agent's ability to replicate and evidence that the agent causes substantial risk of disease to exposed fish populations. This document provides examples of molecularly detected agents causing public concern that were accompanied by little or no data to provide context and assessment of biological significance, highlights important questions to be answered regarding these detections, and provides a suggested pathway of investigative criteria to determine viability and pathogenicity of such agents that are necessary for consideration of any changes to aquatic animal health practices and policies.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kilograms using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 x 10(9) total nucleated cells from mobilized animals and 1.2 x 10(9) total nucleated cells from non-mobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

Transforming Translation Through Quantitative Pharmacology for High-Impact Decision Making in Drug Discovery and Development

Background Patients treated for renal cell carcinoma (RCC) may be diagnosed with a metachronous, contralateral tumor. We evaluated the risks of contralateral tumor development using the Surveillance, Epidemiology, and End Results database. Methods Among RCC patients, we identified those with a metachronous, contralateral RCC diagnosed ≥1 year after primary diagnosis. We performed a competing risks analysis to evaluate associations between clinicopathologic factors and metachronous, bilateral RCC. Cumulative incidence and standardized incidence ratios (SIRs) were calculated. Results There were 80,403 cases of RCC identified, with a median follow-up of 8.3 years; of these, 1063 (1.3%) developed metachronous, contralateral RCC (median of 6 years after diagnosis). The cumulative incidence at 10, 20, and 30 years of follow-up was 1.5%, 3.1%, and 4.7%, respectively. An increased risk was observed among men (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.20-1.55), blacks (HR, 2.00; 95% CI, 1.71-2.33), and those with papillary histology (HR, 1.72; 95% CI, 1.41-2.10). Risk of metachronous disease decreased with increasing age at primary diagnosis (HR per 1-year increase, 0.97; 95% CI, 0.96-0.97). The SIRs were highest among those diagnosed at a younger age and remained elevated even after extended follow-up (>10 years). Conclusions Our findings suggest that the cumulative incidence of metachronous, contralateral RCC may be higher than previously reported. Younger age, black race, papillary histology, and male sex increase the risk of metachronous, contralateral RCC development. The high SIRs seen in all demographic groups may support a rationale for lifelong surveillance, especially in high-risk subgroups with early disease onset.

Author(s): Karuppagounder, Saravanan S; Alin, Lauren; Chen, Yingxin; Brand, David; Bourassa, Megan W; Dietrich, Kristen; Wilkinson, Cassandra M; Nadeau, Colby A; Kumar, Amit; Perry, Steve; Pinto, John T; Darley-Usmar, Victor; Sanchez, Stephanie; Milne, Ginger L; Pratico, Domenico; Holman, Theodore R; Carmichael, S Thomas; Coppola, Giovanni; Colbourne, Frederick; Ratan, Rajiv R | Abstract: ObjectivesN-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke.MethodsHemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC.ResultsNAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ).InterpretationNAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

Objective To determine indications, surgical efficacy, and audiologic outcomes of replacing Advanced Bionics Clarion C1.2 internal devices (Advanced Bionics, LLC, Valencia, CA) as a means of technology upgrade. Study design Retrospective review, case series. Methods Ten patients were initially implanted as a child (mean age = 3.87 years) and underwent cochlear implant reimplantation (CIR) with current Advanced Bionics internal device as a young adult (mean duration of implant use = 15.66 years). Demographic data and pre- and post-CIR speech perception scores were collected. Results Technology upgrade was the primary (9) or secondary (1) motivation for CIR. No surgical complications were noted, and full insertion was obtained in nine cases. Intraoperative impedance levels and neural response imaging measures were within normal limits for eight patients. At most recent post-CIR follow-up evaluation, all patients (100%) performed within or better than the 95% confidence interval of their pre-CIR word and sentence recognition scores; and 55.6%, 50.0%, and 50.0% of patients performed above the 95% confidence interval of their pre-CIR scores for the CNC words, sentences in quiet, and sentences in noise, respectively. Conclusion Post-CIR audiological benefit was stable or improved compared to pre-CIR results in all categories by 3 months after reactivation. Given these results, patients who are unable to use the most current external processors due to incompatibility with a legacy internal device could consider reimplanation to optimize their overall performance with a cochlear implant. Level of evidence 4 Laryngoscope, 129:748-753, 2019.

Incidence rates are an important population-level disease risk measure. Cancer incidence data in the United States, which are collected by disease registries, have been spatiotemporally sparse. Back-calculation methods can yield incidence estimates for a spatial domain by solving a convolution equation that relates mortality to incidence through survival estimates. We propose a novel back-calculation approach that uses spatiotemporal age-period-cohort (APC) modeling to estimate incidence for spatial units within a region. The method is applied to state-specific lung cancer data in the United States for males ages 30 to 84 in years 1975-2004. SEER data are used to model cancer progression from incidence to mortality along three timescales (APC) and across four regions. Using mortality data from National Vital Statistics System, incidence is back-calculated for unique birth cohorts in 49 states. A conditionally autoregressive model with cubic splines for temporal effects is used to smooth back-calculated estimates. Bayesian estimates of model parameters are obtained using integrated nested Laplace approximation. Results show varying time trends in lung cancer risk across states, which may quantify effects of state policies.

Objective To characterize current vestibular schwannoma (VS) surgery outcomes with a nationwide database and identify factors associated with increased complications and prolonged hospital course. Study Design Retrospective review utilizing the University HealthSystem Consortium national inpatient database. Setting US academic health centers. Subjects and Methods Data from patients undergoing VS surgery were analyzed over a 3-year time span (October 2012 to September 2015). Surgical outcomes, such as length of stay (LOS), complications, and mortality, were analyzed on the basis of race, sex, age, and comorbidities during the 30-day postoperative period. Results A total of 3697 VS surgical cases were identified. The overall mortality rate was 0.38%, and the overall complication rate was 5.3%. Advanced age significantly affected intensive care unit LOS, mortality, and complications ( P = .04). Comorbidities, including hypertension, obesity, and depression, also significantly increased complication rates ( P = .02). Sixty-eight patients (1.8%) had a history of irradiation, and they had a significantly increased LOS ( P = .03). Conclusion Modern VS surgery has a low mortality rate and a relatively low rate of complications. Several factors contribute to high complication rates, including age and comorbidities. These data will help providers in counseling patients on which treatment course might be best suited for them.

The Lower Keys marsh rabbit (Sylvilagus palustris hefneri) is one of many endangered endemic species of the Florida Keys. The main threats are habitat loss and fragmentation from sea‐level rise, development, and habitat succession. Exotic predators such as free‐ranging domestic cats (Felis catus) pose an additional threat to these endangered small mammals. Management strategies have focused on habitat restoration and exotic predator control. However, the effectiveness of predator removal and the effects of anthropogenic habitat modifications and restoration have not been evaluated. Between 2013 and 2015, we used camera traps to survey marsh rabbits and free‐ranging cats at 84 sites in the National Key Deer Refuge, Big Pine Key, Florida, USA. We used dynamic occupancy models to determine factors associated with marsh rabbit occurrence, colonization, extinction, and the co‐occurrence of marsh rabbits and cats during a period of predator removal. Rabbit occurrence was positively related to freshwater habitat and patch size, but was negatively related to the number of individual cats detected at each site. Furthermore, marsh rabbit colonization was negatively associated with relative increases in the number of individual cats at each site between survey years. Cat occurrence was negatively associated with increasing distance from human developments. The probability of cat site extinction was positively related to a 2‐year trapping effort, indicating that predator removal reduced the cat population. Dynamic co‐occurrence models suggested that cats and marsh rabbits co‐occur less frequently than expected under random conditions, whereas co‐detections were site and survey‐specific. Rabbit site extinction and colonization were not strongly conditional on cat presence, but corresponded with a negative association. Our results suggest that while rabbits can colonize and persist at sites where cats occur, it is the number of individual cats at a site that more strongly influences rabbit occupancy and colonization. These findings indicate that continued predator management would likely benefit endangered small mammals as they recolonize restored habitats.

OBJECTIVE Identify factors associated with benefit of middle ear implants (MEIs) as compared to conventional hearing aids (HAs). STUDY DESIGN Independent review of audiological data from a multicenter prospective U.S. Food and Drug Administration (FDA) clinical trial. Preoperative and postoperative earphone, unaided/aided/implanted pure-tone thresholds, and word recognition scores were evaluated. RESULTS Ninety-one subjects were included in this study. Mean word recognition was better with MEIs than with HAs (81.8% ± 12.0% vs. 77.6% ± 14.6%, P = 0.035). Word recognition with MEIs showed a low positive correlation with word recognition measured with earphones (r = 0.25, P = 0.016) and a moderate positive correlation with aided word recognition (r = 0.42, P

Objectives Determine the impact of cochlear implantation on quality of life (QOL) and determine the correlation between QOL and speech recognition ability. Study Design Two authors independently searched PubMed, Medline, Scopus, and the Cumulative Index to Nursing and Allied Health Literature to identify studies reporting hearing-specific or cochlear implant (CI)–specific QOL outcomes before and after cochlear implantation, and studies reporting correlations between QOL and speech recognition after cochlear implantation. Data from the included articles were obtained independently by two authors. Standardized mean difference (SMD) for each measure and pooled effects were determined to assess improvement in QOL before and after cochlear implantation. Results From 14 articles with 679 CI patients who met the inclusion criteria, pooled analyses of all hearing-specific QOL measures revealed a very strong improvement in QOL after cochlear implantation (SMD = 1.77). Subset analysis of CI-specific QOL measures also showed very strong improvement (SMD = 1.69). Thirteen articles with 715 patients met the criteria to evaluate associations between QOL and speech recognition. Pooled analyses showed a low positive correlation between hearing-specific QOL and word recognition in quiet (r = 0.213), sentence recognition in quiet (r = 0.241), and sentence recognition in noise (r = 0.238). Subset analysis of CI-specific QOL showed similarly low positive correlations with word recognition in quiet (r = 0.213), word recognition in noise (r = 0.241), and sentence recognition in noise (r = 0.255). Conclusions Using hearing-specific and CI-specific measures of QOL, patients report significantly improved QOL after cochlear implantation. However, widely used clinical measures of speech recognition are poor predictors of patient-reported QOL with CIs. Laryngoscope, 2017

Our objective was to prospectively evaluate implementation of a new cochlear implant (CI) mapping technique, image-guided cochlear implant programming (IGCIP), at a site distant to the site of development. IGCIP consists of identifying the geometric relationship between CI electrodes and the modiolus and deactivating electrodes that interfere with neighboring electrodes. IGCIP maps for 17 ears of 15 adult CI patients were developed at a central image-processing center, Vanderbilt, and implemented at a distant tertiary care center, House Ear Institute. Before IGCIP and again 4 weeks after, qualitative and quantitative measures were made. While there were no statistically significant groupwise differences detected between baseline and IGCIP qualitative or quantitative measures, 11 of the 17 (64.7%) elected to keep the IGCIP map. Computed tomography (CT) image quality appears to be crucial for successful IGCIP, with 100% of those with high-resolution CT scans keeping their maps compared to 53.8% without.

We reviewed the 2020 American College of Rheumatology (ACR) Guideline for the Management of Gout with interest. First, we want to commend the committee for the excellent job they did in updating the guidelines. We noted the commitment to evidence- based medicine and also that the indications for starting urate-lowering therapy (ULT) have been expanded to consider patients with infrequent gout flares or a first gout flare if they also have moderate to severe chronic kidney disease (CKD stage ≥ 3), marked hyperuricemia (serum urate (SU) > 9 mg/dl) or kidney stones.

Tert-butyloxycarbonyl (t-Boc)-based native chemical ligation (NCL) techniques commonly employ hydrogen fluoride (HF) to create the thioester fragment required for the ligation process. Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin-I (HwTx-I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx-I has an X-Cys(16) -Cys(17) -X sequence mid-region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 μL with a 0.5-h incubation on ice, followed by 2.0 h at room temperature. RP-HPLC/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx-I N-terminal thioester fragment (Ala(1) -Cys(16) ), which was used in preparation for NCL. The resulting chemically ligated HwTx-I was oxidized/folded, purified, and then assessed for pharmacological target selectivity. Native-like HwTx-I produced by this method yielded an EC50 value of 340.5 ± 26.8 nM for Nav 1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav 1.3, this being similar to previous literature results using native material. This article represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 737-745, 2016.

As human populations and associated development increase, interactions between humans and wildlife are occurring with greater frequency. The effects of these interactions, particularly on species whose populations are declining, are of great interest to ecologists, conservationists, land managers and natural resource policy-makers. The American Oystercatcher Haematopus palliatus, a species of conservation concern in the USA, nests on coastal beaches subject to various forms of anthropogenic disturbance, including aircraft overflights, off-road vehicles and pedestrians. This study assessed the effects of these human disturbances on the incubation behaviour and reproductive success of nesting American Oystercatchers at Cape Lookout National Seashore, on the Atlantic coast of the USA. We expanded on-going monitoring of Oystercatchers at Cape Lookout National Seashore by supplementing periodic visual observations with continuous 24-h video and audio recording at nests. Aircraft overflights were not associated with changes in Oystercatcher incubation behaviour, and we found no evidence that aircraft overflights influenced Oystercatcher reproductive success. However, Oystercatchers were on their nests significantly less often during off-road vehicle and pedestrian events than they were during control periods before the events, and an increase in the number of off-road vehicles passing a nest during incubation was consistently associated with significant reductions in daily nest survival (6% decrease in daily nest survival for a one-vehicle increase in the average number of vehicles passing a nest each day; odds ratio = 0.94; 95% confidence interval (CI) 0.90, 0.98) and hatching success (12% decrease in hatching success for a one-vehicle increase in the average number of vehicles passing a nest each day; odds ratio = 0.88; 95% CI 0.76, 0.97). Management of vehicles and pedestrians in areas of Oystercatcher breeding is important for the conservation of American Oystercatchers.