The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent.

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

Effect of aspirin response signature gene expression on preterm birth and preeclampsia among women with lupus: a pilot study.

BACKGROUND: Women with lupus have an increased risk of preeclampsia and preterm birth, and aspirin 81 mg/day is recommended as a preventative measure for preeclampsia. This pilot study quantified the association between a 60-gene aspirin response signature (ARS) gene expression with preterm birth and preeclampsia risk among women with lupus taking aspirin. METHODS: The analysis included 48 RNA samples from 23 pregnancies in the Duke Autoimmunity Pregnancy Registry. RNA was isolated from peripheral blood, and quantitative polymerase chain reaction was performed for ARS genes. The primary outcome was poor pregnancy outcome (preeclampsia or preterm birth). Gene expression was modeled as a response to presence or absence of a poor pregnancy outcome using linear regression models, stratified by trimester. RESULTS: Of the 23 pregnancies, nine delivered preterm and four had preeclampsia. Expression of PBX1 and MMD was higher in the second trimester among patients who experienced a poor pregnancy outcome compared to those who did not. However, in a global test of all ARS genes, we identified no association between expression of ARS genes and poor pregnancy outcomes. CONCLUSION: Our pilot study identified two candidate genes that are reflective of the platelet function response to aspirin. Further work is needed to determine the role of these genes in identifying women with lupus at high risk for preeclampsia and preterm delivery despite aspirin therapy.

Systematic reanalysis of genomic data improves quality of variant interpretation.

As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize the benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants that were not initially reported in 23 individuals, 6 described here, comprising a 16% increase in P/LP yield. We also downgraded 3 LP and 6 variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after 3 years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.

Giant spin Seebeck effect in a non-magnetic material.

The spin Seebeck effect is observed when a thermal gradient applied to a spin-polarized material leads to a spatially varying transverse spin current in an adjacent non-spin-polarized material, where it gets converted into a measurable voltage. It has been previously observed with a magnitude of microvolts per kelvin in magnetically ordered materials, ferromagnetic metals, semiconductors and insulators. Here we describe a signal in a non-magnetic semiconductor (InSb) that has the hallmarks of being produced by the spin Seebeck effect, but is three orders of magnitude larger (millivolts per kelvin). We refer to the phenomenon that produces it as the giant spin Seebeck effect. Quantizing magnetic fields spin-polarize conduction electrons in semiconductors by means of Zeeman splitting, which spin-orbit coupling amplifies by a factor of ∼25 in InSb. We propose that the giant spin Seebeck effect is mediated by phonon-electron drag, which changes the electrons' momentum and directly modifies the spin-splitting energy through spin-orbit interactions. Owing to the simultaneously strong phonon-electron drag and spin-orbit coupling in InSb, the magnitude of the giant spin Seebeck voltage is comparable to the largest known classical thermopower values.

Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis.

BACKGROUND: Distant metastasis accounts for 90% of deaths from colorectal cancer (CRC). Genomic heterogeneity has been reported in various solid malignancies, but remains largely under-explored in metastatic CRC tumors, especially in primary to metastatic tumor evolution. PATIENTS AND METHODS: We conducted high-depth whole-exome sequencing in multiple regions of matched primary and metastatic CRC tumors. Using a total of 28 tumor, normal, and lymph node tissues, we analyzed inter- and intra-individual heterogeneity, inferred the tumor subclonal architectures, and depicted the subclonal evolutionary routes from primary to metastatic tumors. RESULTS: CRC has significant inter-individual but relatively limited intra-individual heterogeneity. Genomic landscapes were more similar within primary, metastatic, or lymph node tumors than across these types. Metastatic tumors exhibited less intratumor heterogeneity than primary tumors, indicating that single-region sequencing may be adequate to identify important metastasis mutations to guide treatment. Remarkably, all metastatic tumors inherited multiple genetically distinct subclones from primary tumors, supporting a possible polyclonal seeding mechanism for metastasis. Analysis of one patient with the trio samples of primary, metastatic, and lymph node tumors supported a mechanism of synchronous parallel dissemination from the primary to metastatic tumors that was not mediated through lymph nodes. CONCLUSIONS: In CRC, metastatic tumors have different but less heterogeneous genomic landscapes than primary tumors. It is possible that CRC metastasis is, at least partly, mediated through a polyclonal seeding mechanism. These findings demonstrated the rationale and feasibility for identifying and targeting primary tumor-derived metastasis-potent subclones for the prediction, prevention, and treatment of CRC metastasis.

Genetic associations with viral respiratory illnesses and asthma control in children.

BACKGROUND: Viral respiratory infections can cause acute wheezing illnesses in children and exacerbations of asthma. OBJECTIVE: We sought to identify variation in genes with known antiviral and pro-inflammatory functions to identify specific associations with more severe viral respiratory illnesses and the risk of virus-induced exacerbations during the peak fall season. METHODS: The associations between genetic variation at 326 SNPs in 63 candidate genes and 10 phenotypes related to viral respiratory infection and asthma control were examined in 226 children enrolled in the RhinoGen study. Replication of asthma control phenotypes was performed in 2128 children in the Copenhagen Prospective Study on Asthma in Childhood (COPSAC). Significant associations in RhinoGen were further validated using virus-induced wheezing illness and asthma phenotypes in an independent sample of 122 children enrolled in the Childhood Origins of Asthma (COAST) birth cohort study. RESULTS: A significant excess of P values smaller than 0.05 was observed in the analysis of the 10 RhinoGen phenotypes. Polymorphisms in 12 genes were significantly associated with variation in the four phenotypes showing a significant enrichment of small P values. Six of those genes (STAT4, JAK2, MX1, VDR, DDX58, and EIF2AK2) also showed significant associations with asthma exacerbations in the COPSAC study or with asthma or virus-induced wheezing phenotypes in the COAST study. CONCLUSIONS: We identified genetic factors contributing to individual differences in childhood viral respiratory illnesses and virus-induced exacerbations of asthma. Defining mechanisms of these associations may provide insight into the pathogenesis of viral respiratory infections and virus-induced exacerbations of asthma.

Clinical course of 161 untreated and tenofovir-treated chronic hepatitis B pregnant patients in a low hepatitis B virus endemic region.

Hepatitis B immunoprophylaxis failure is linked to high maternal viraemia. There is limited North American data on hepatitis B outcomes in pregnancy. Pregnant hepatitis B carriers were enrolled January 2011-December 2014 and offered tenofovir in the 3rd trimester if hepatitis B virus (HBV)-DNA was >7-log IU/mL. Outcomes were determined in treated vs untreated patients. In total, 161 women with 169 pregnancies (one twin, 170 infants; median age 32 years), 18% (29/161) HBeAg+ and median HBV-DNA 2.51 log IU/mL (IQR 1.66-3.65; range 0.8-8.1) were studied. 14.3% (23/161) received tenofovir due to high viral load (16/23, median 74 days, IQR 59-110) or due to liver disease (7/23). In 10/16 treated due to high viraemia, with confirmed adherence, follow-up HBV-DNA showed a 5.49 log decline (P = 0.003). In treatment naïve mothers, median alanine aminotransferase (ALT) increased from 17 IU/L (IQR 12-24) to 29 (IQR 18-36) post-partum (P = 1.5e-7). In seven highly viraemic mothers who declined therapy (HBV-DNA >8-log IU/mL); median ALT increased ~3X from baseline (P < 0.01). 26% (44/169) had Caesarean section with no difference in treated vs untreated subjects. No tenofovir-treated mothers had renal dysfunction. Data were available on 167/170 infants; in 50.8% (85/167) who completed immunoprophylaxis, 98.8% (84/85, including 12 exposed to tenofovir in utero) were HBV immune. One infant born to an HBeAg+ mother with HBV-DNA >8-log IU/mL failed immunoprophylaxis. In this prospective Canadian cohort study, most untreated mothers experienced mild HBV flares. Tenofovir in pregnancy is well tolerated and reduces viral load prior to parturition.

Experimental Demonstration of the Collisionless Plasmoid Instability below the Ion Kinetic Scale during Magnetic Reconnection.

The spontaneous formation of magnetic islands is observed in driven, antiparallel magnetic reconnection on the Terrestrial Reconnection Experiment. We here provide direct experimental evidence that the plasmoid instability is active at the electron scale inside the ion diffusion region in a low collisional regime. The experiments show the island formation occurs at a smaller system size than predicted by extended magnetohydrodynamics or fully collisionless simulations. This more effective seeding of magnetic islands emphasizes their importance to reconnection in naturally occurring 3D plasmas.

Acceptability and Effectiveness of Assisted Human Immunodeficiency Virus Partner Services in Mozambique: Results From a Pilot Program in a Public, Urban Clinic.

BACKGROUND: Assisted partner services (APS) involves offering persons with human immunodeficiency virus (HIV) assistance notifying and testing their sex partners. Assisted partner services is rarely available in sub-Saharan Africa. We instituted a pilot APS program in Maputo, Mozambique. METHODS: Between June and September 2014, community health workers (CHWs) offered APS to persons with newly diagnosed HIV (index patients [IPs]). Community health workers interviewed IPs at baseline, 4 and 8 weeks. At baseline, CHWs counseled IPs to notify partners and encourage their HIV testing, but did not notify partners directly. At 4 weeks, CHWs notified partners directly. We compared 4- and 8-week outcomes to estimate the impact of APS on partner notification, HIV testing and HIV case finding. RESULTS: Community health workers offered 223 IPs APS, of whom 220 (99%) accepted; CHWs collected complete follow-up data on 206 persons; 79% were women, 74% were married, and 50% named >1 sex partner. Index patients named 262 HIV-negative partners at baseline. At 4 weeks, before APS, IPs had notified 193 partners (74%), but only 82 (31%) had HIV tested; 43 (13%) tested HIV positive. Assisted partner services resulted in the notification of 22 additional partners, testing of 83 partners and 43 new HIV diagnoses. In relative terms, APS increased partner notification, testing, and HIV case finding by 13%, 101%, and 125%. Seventy-two (35%) of 206 IPs were in ongoing HIV serodiscordant partnerships. Only 2.5 IPs needed to receive APS to identify a previously undiagnosed HIV-infected partner or an ongoing HIV serodiscordant partnership. Two (1%) IPs reported APS-related adverse events. CONCLUSIONS: Assisted partner services is acceptable to Mozambicans newly diagnosed with HIV, identifies large numbers of serodiscordant partnerships and persons with undiagnosed HIV, and poses a low risk of adverse events.

Circadian dysregulation of clock genes: clues to rapid treatments in major depressive disorder.

Conventional antidepressants require 2-8 weeks for a full clinical response. In contrast, two rapidly acting antidepressant interventions, low-dose ketamine and sleep deprivation (SD) therapy, act within hours to robustly decrease depressive symptoms in a subgroup of major depressive disorder (MDD) patients. Evidence that MDD may be a circadian-related illness is based, in part, on a large set of clinical data showing that diurnal rhythmicity (sleep, temperature, mood and hormone secretion) is altered during depressive episodes. In a microarray study, we observed widespread changes in cyclic gene expression in six regions of postmortem brain tissue of depressed patients matched with controls for time-of-death (TOD). We screened 12 000 transcripts and observed that the core clock genes, essential for controlling virtually all rhythms in the body, showed robust 24-h sinusoidal expression patterns in six brain regions in control subjects. In MDD patients matched for TOD with controls, the expression patterns of the clock genes in brain were significantly dysregulated. Some of the most robust changes were seen in anterior cingulate (ACC). These findings suggest that in addition to structural abnormalities, lesion studies, and the large body of functional brain imaging studies reporting increased activation in the ACC of depressed patients who respond to a wide range of therapies, there may be a circadian dysregulation in clock gene expression in a subgroup of MDDs. Here, we review human, animal and neuronal cell culture data suggesting that both low-dose ketamine and SD can modulate circadian rhythms. We hypothesize that the rapid antidepressant actions of ketamine and SD may act, in part, to reset abnormal clock genes in MDD to restore and stabilize circadian rhythmicity. Conversely, clinical relapse may reflect a desynchronization of the clock, indicative of a reactivation of abnormal clock gene function. Future work could involve identifying specific small molecules capable of resetting and stabilizing clock genes to evaluate if they can rapidly relieve symptoms and sustain improvement.

Optical control of internal electric fields in band gap-graded InGaN nanowires.

InGaN nanowires are suitable building blocks for many future optoelectronic devices. We show that a linear grading of the indium content along the nanowire axis from GaN to InN introduces an internal electric field evoking a photocurrent. Consistent with quantitative band structure simulations we observe a sign change in the measured photocurrent as a function of photon flux. This negative differential photocurrent opens the path to a new type of nanowire-based photodetector. We demonstrate that the photocurrent response of the nanowires is as fast as 1.5 ps.

Long-term transmission of measles virus in Central and continental Western Europe.

The World Health Organization (WHO) has adopted an elimination goal for measles and rubella, which is supposed to be met in the WHO European Region (EUR) by 2015. For verification of elimination, it is required that the genotyping data of detected measles viruses provide evidence for the interruption of endemic transmission. In order to record and assess the extent of endemic measles virus (MV) circulation in a part of the EUR, we analyzed transmission chains of the epidemiologically most relevant MV variants identified in Central and continental Western Europe (CCWE) from 2006 to 2013. Based on MV sequence data deposited in the WHO global database for molecular surveillance of measles (MeaNS), the circulation period was calculated for each MV variant at the country-level and for the entire region of CCWE. The MV variants "D5-Okinawa," "D4-Hamburg," "D4-Manchester," and "D8-Frankfurt-Main" spread widely in CCWE; they caused large and long-lasting outbreaks with secondary spread that resulted in additional outbreaks. Nation-wide outbreaks (epidemics) with thousands of measles cases occurred in four countries (Switzerland, France, Bulgaria, and Romania) and were characterized by continuous detection of the same MV variant for more than 12 months suggesting endemic transmission. In the entire region of CCWE, the circulation period of the four predominant MV variants ranged from 18 to 44 months. The long-lasting MV transmission which affected predominantly unvaccinated individuals in different hard-to-reach groups and in the general population is not consistent with the measles elimination goal. Additional efforts are necessary to meet the elimination target in the EUR.

Genome sequence analysis of Ebola virus in clinical samples from three British healthcare workers, August 2014 to March 2015.

We determined complete viral genome sequences from three British healthcare workers infected with Ebola virus (EBOV) in Sierra Leone, directly from clinical samples. These sequences closely resemble those previously observed in the current Ebola virus disease outbreak in West Africa, with glycoprotein and polymerase genes showing the most sequence variation. Our data indicate that current PCR diagnostic assays remain suitable for detection of EBOV in this epidemic and provide confidence for their continued use in diagnosis.

The present and future disease burden of hepatitis C virus (HCV) infection with today's treatment paradigm.

The disease burden of hepatitis C virus (HCV) is expected to increase as the infected population ages. A modelling approach was used to estimate the total number of viremic infections, diagnosed, treated and new infections in 2013. In addition, the model was used to estimate the change in the total number of HCV infections, the disease progression and mortality in 2013-2030. Finally, expert panel consensus was used to capture current treatment practices in each country. Using today's treatment paradigm, the total number of HCV infections is projected to decline or remain flat in all countries studied. However, in the same time period, the number of individuals with late-stage liver disease is projected to increase. This study concluded that the current treatment rate and efficacy are not sufficient to manage the disease burden of HCV. Thus, alternative strategies are required to keep the number of HCV individuals with advanced liver disease and liver-related deaths from increasing.

Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses.

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m(-2) was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m(-2) should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation.

Early events in neuronal differentiation are generally considered to be regulated by factors independent of alterations in membrane permeability. Weaver mice harbour a mutation that blocks neuronal differentiation just after cessation of cell division, prior to cell migration and synaptogenesis. Cerebellar granule cells in homozygous weaver mice fail to differentiate, either because intrinsic cues are absent or because the granule cells are unable to respond to those cues. We now report that weaver mice have a missense mutation in a gene encoding a G-protein coupled inward rectifier potassium channel. The mutation alters the putative ion-permeable, pore-forming domain of the protein, suggesting that granule cell differentiation is regulated by changes in membrane permeability.

The Huntington's disease candidate region exhibits many different haplotypes.

Analysis of 78 Huntington's disease (HD) chromosomes with multi-allele markers revealed 26 different haplotypes, suggesting a variety of independent HD mutations. The most frequent haplotype, accounting for about one third of disease chromosomes, suggests that the disease gene is between D4S182 and D4S180. However, the paucity of an expected class of chromosomes that can be related to this major haplotype by assuming single crossovers may reflect the operation of other mechanisms in creating haplotype diversity. Some of these mechanisms sustain alternative scenarios that do not require a multiple mutational origin for HD and/or its positioning between D4S182 and D4S180.

Trinucleotide repeat length instability and age of onset in Huntington's disease.

The initial observation of an expanded and unstable trinucleotide repeat in the Huntington's disease gene has now been confirmed and extended in 150 independent Huntington's disease families. HD chromosomes contained 37-86 repeat units, whereas normal chromosomes displayed 11-34 repeats. The HD repeat length was inversely correlated with the age of onset of the disorder. The HD repeat was unstable in more than 80% of meiotic transmissions showing both increases and decreases in size with the largest increases occurring in paternal transmissions. The targeting of spermatogenesis as a particular source of repeat instability is reflected in the repeat distribution of HD sperm DNA. The analysis of the length and instability of individual repeats in members of these families has profound implications for presymptomatic diagnosis.

Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice.

Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown. We found that mice lacking cystatin B develop myoclonic seizures and ataxia, similar to symptoms seen in the human disease. The principal cytopathology appears to be a loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA and other cellular changes characteristic of apoptosis. This mouse model of EPM1 provides evidence that cystatin B, a non-caspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.

De novo expansion of a (CAG)n repeat in sporadic Huntington's disease.

Huntington's disease (HD) chromosomes contain an expanded unstable (CAG)n repeat in chromosome 4p16.3. We have examined nine families with potential de novo expression of the disease. With one exception, all of the affected individuals had 42 or more repeat units, well above the normal range. In four families, elderly unaffected relatives inherited the same chromosome as that containing the expanded repeat in the proband, but had repeat lengths of 34-38 units, spanning the gap between the normal and HD distributions. Thus, mutation to HD is usually associated with an expansion from an already large repeat.