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.

Hospitalisation and mortality risk of SARS-COV-2 variant omicron sub-lineage BA.2 compared to BA.1 in England.

The Omicron variant of SARS-CoV-2 became the globally dominant variant in early 2022. A sub-lineage of the Omicron variant (BA.2) was identified in England in January 2022. Here, we investigated hospitalisation and mortality risks of COVID-19 cases with the Omicron sub-lineage BA.2 (n = 258,875) compared to BA.1 (n = 984,337) in a large cohort study in England. We estimated the risk of hospital attendance, hospital admission or death using multivariable stratified proportional hazards regression models. After adjustment for confounders, BA.2 cases had lower or similar risks of death (HR = 0.80, 95% CI 0.71-0.90), hospital admission (HR = 0.88, 95% CI 0.83-0.94) and any hospital attendance (HR = 0.98, 95% CI 0.95-1.01). These findings that the risk of severe outcomes following infection with BA.2 SARS-CoV-2 was slightly lower or equivalent to the BA.1 sub-lineage can inform public health strategies in countries where BA.2 is spreading.

Reduction of the Carbapenemase Inactivation Method (CIM) assay time by real-time PCR.

Carbapenemase Inactivation Method (CIM) is a test to detect presence of the carbapenemase in Gram-negative bacteria. Determination of the carbapenemase production by inactivation of meropenem requires that a zone of control E. coli inhibition be measured approximately 6-24 h after plating. We have modified the CIM test by developing a rapid method which instead measures the growth of E. coli indicator strain ATCC 25922 using real-time PCR, referred to as a nucleic acid testing CIM (natCIM). Our natCIM, therefore reduces the detecting time from 6 to 24 h to approximately 4 h.

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.

Electrophysiological evaluation of extracellular spermine and alkaline pH on synaptic human GABAA receptors.

Polyamines have fundamental roles in brain homeostasis as key modulators of cellular excitability. Several studies have suggested alterations in polyamine metabolism in stress related disorders, suicide, depression, and neurodegeneration, making the pharmacological modulation of polyamines a highly appealing therapeutic strategy. Polyamines are small aliphatic molecules that can modulate cationic channels involved in neuronal excitability. Previous indirect evidence has suggested that polyamines can modulate anionic GABAA receptors (GABAARs), which mediate inhibitory signaling and provide a direct route to reduce hyperexcitability. Here, we attempted to characterize the effect that spermine, the polyamine with the strongest reported effect on GABAARs, has on human postmortem native GABAARs. We microtransplanted human synaptic membranes from the dorsolateral prefrontal cortex of four cases with no history of mental or neurological disorders, and directly recorded spermine effects on ionic GABAARs responses on microtransplanted oocytes. We show that in human synapses, inhibition of GABAARs by spermine was better explained by alkalization of the extracellular solution. Additionally, spermine had no effect on the potentiation of GABA-currents by diazepam, indicating that even if diazepam binding is enhanced by spermine, it does not translate to changes in functional activity. Our results clearly demonstrate that while extracellular spermine does not have direct effects on human native synaptic GABAARs, spermine-mediated shifts of pH inhibit GABAARs. Potential spermine-mediated increase of pH in synapses in vivo may therefore participate in increased neuronal activity observed during physiological and pathological states, and during metabolic alterations that increase the release of spermine to the extracellular milieu.

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.

Impact of controlled attenuation parameter on detecting fibrosis using liver stiffness measurement.

BACKGROUND: Liver fibrosis is often accompanied by steatosis, particularly in patients with non-alcoholic fatty liver disease (NAFLD), and its non-invasive characterisation is of utmost importance. Vibration-controlled transient elastography is the non-invasive method of choice; however, recent research suggests that steatosis may influence its diagnostic performance. Controlled Attenuation Parameter (CAP) added to transient elastography enables simultaneous assessment of steatosis and fibrosis. AIM: To determine how to use CAP in interpreting liver stiffness measurements. METHODS: This is a secondary analysis of data from an individual patient data meta-analysis on CAP. The main exclusion criteria for the current analysis were unknown aetiology, unreliable elastography measurement and data already used for the same research question. Aetiology-specific liver stiffness measurement cut-offs were determined and used to estimate positive and negative predictive values (PPV/NPV) with logistic regression as functions of CAP. RESULTS: Two thousand and fifty eight patients fulfilled the inclusion criteria (37% women, 18% NAFLD/NASH, 42% HBV, 40% HCV, 51% significant fibrosis ≥ F2). Youden optimised cut-offs were only sufficient for ruling out cirrhosis (NPV of 98%). With sensitivity and specificity-optimised cut-offs, NPV for ruling out significant fibrosis was moderate (70%) and could be improved slightly through consideration of CAP. PPV for significant fibrosis and cirrhosis were 68% and 55% respectively, despite specificity-optimised cut-offs for cirrhosis. CONCLUSIONS: Liver stiffness measurement values below aetiology-specific cut-offs are very useful for ruling out cirrhosis, and to a lesser extent for ruling out significant fibrosis. In the case of the latter, Controlled Attenuation Parameter can improve interpretation slightly. Even if cut-offs are very high, liver stiffness measurements are not very reliable for ruling in fibrosis or cirrhosis.

Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations.

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

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.

Tenofovir disoproxil fumarate significantly decreases serum lipoprotein levels compared with entecavir nucleos(t)ide analogue therapy in chronic hepatitis B carriers.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) and entecavir (ETV) are first-line treatments for chronic hepatitis B (CHB). Studies suggest lipid lowering effect of TDF in human immunodeficiency virus positive (HIV+) individuals, but the effect on lipids and cardiovascular disease (CVD) risk in CHB is unknown. AIM: To compare TDF vs ETV effects on lipid levels in CHB. METHODS: In this retrospective cohort study, data on serum lipids and CVD risk factors at baseline and ~1 year on TDF or ETV were collected from CHB carriers. We used propensity score matched models to assess the effect on total cholesterol (TC), LDL-C, HDL and triglycerides (TGL). RESULTS: In 348 patients, median age was 57 (IQR: 47-65 years), 63% were male, 77% were Asian, 19% were cirrhotic, 25% were HBeAg positive at baseline, and 72% received TDF vs 28% ETV. ETV-treated patients were older (median age: 60 vs 55, P<.01), had similar smoking and hypertension rates, but diabetes and dyslipidemia were more prevalent (19% vs 9%, P=.01; 14% vs 6%, P=.05, respectively). In propensity score matched models for age, gender, usage of lipid lowering agents, dyslipidemia and diabetes, TDF-treated patients were more likely to show a 20% decrease in TC (95% CI: 3%-25%), LDL-C (95% CI: 1%-25%) and HDL-C (CI: 10%-30%) levels compared with those on ETV. No change in TGL was observed in either group. CONCLUSIONS: A greater decline in TC, LDL-C and HDL was observed in CHB carriers receiving TDF compared with ETV. These data may influence anti-viral choice in CHB carriers at risk for CVD.

Molecular surveillance of measles and rubella in the WHO European Region: new challenges in the elimination phase.

BACKGROUND: The WHO European Region (EUR) has adopted the goal of eliminating measles and rubella but individual countries perform differently in achieving this goal. Measles virus spread across the EUR by mobile groups has recently led to large outbreaks in the insufficiently vaccinated resident population. As an instrument for monitoring the elimination process and verifying the interruption of endemic virus transmission, molecular surveillance has to provide valid and representative data. Irrespective of the country's specific situation, it is required to ensure the functionality of the laboratory surveillance that is supported by the WHO Global Measles and Rubella Laboratory Network. AIMS: To investigate whether the molecular surveillance in the EUR is adequate for the challenges in the elimination phase, we addressed the quality assurance of molecular data, the continuity and intensity of molecular monitoring, and the analysis of transmission chains. SOURCES: Published articles, the molecular External Quality Assessment Programme of the WHO, the Centralized Information System for Infectious Diseases of the WHO EUR and the WHO Measles and Rubella Nucleotide Surveillance databases served as information sources. CONTENT: Molecular proficiency testing conducted by the WHO in 2016 has shown that the expertise for measles and rubella virus genotyping exists in all parts of the EUR. The analysis of surveillance data reported nationally to the WHO in 2013-2016 has revealed some countries with outbreaks but not sufficiently representative molecular data. Long-lasting supranational MV transmission chains were identified. IMPLICATIONS: A more systematic molecular monitoring and recording of the transmission pattern for the whole EUR could help to create a meaningful picture of the elimination process.

Neutrophil to lymphocyte ratio associated with prognosis of lung cancer.

PURPOSE: Many studies recently focus on complicated and expensive genomic tests, but the prognostic values of biochemical markers which are easily obtained in clinics are largely overlooked and without further exploration. This study assesses the association of neutrophil-lymphocyte-ratio (NLR) with prognosis of lung cancer patients. METHODS: In 1032 patients with histologically confirmed lung cancer, the association of pretreatment NLR values with overall survival (OS) was evaluated using a Cox proportional hazards model and the temporal relationship of longitudinal NLR was assessed using a mixed effects model. RESULTS: Compared to the patients with a low pretreatment NLR value, those with elevated NLR exhibited a statistically significant worse OS with a hazard ratio (HR) of 1.50 (P < 0.0001) after adjusting for age, gender, race, smoking status, drinking status, tumor stage, tumor grade, histology, and treatments. A significant trend of increasing HRs along with increasing NLR values was observed. The increased risk of death conferred by pretreatment NLR values reached a peak level around 2 years after diagnosis. Moreover, in longitudinal analysis, we observed a trend of dramatically increased NLR values in patients who died during follow-up, but stable NLR values in those who were still alive, with a significant interaction of death-alive status with follow-up time (P < 0.0001). CONCLUSIONS: Elevated NLR is a potential biomarker to identify lung cancer patients with poor prognosis and should be validated in a future clinical trial.

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.

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.

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.

Variable telomere length across post-mortem human brain regions and specific reduction in the hippocampus of major depressive disorder.

Stress can be a predisposing factor to psychiatric disorders and has been associated with decreased neurogenesis and reduced hippocampal volume especially in depression. Similarly, in white blood cells chronic psychological stress has been associated with telomere shortening and with mood disorders and schizophrenia (SZ). However, in previous post-mortem brain studies from occipital cortex and cerebellum, no difference in telomere length was observed in depression. We hypothesized that in psychiatric disorders, stress-driven accelerated cellular aging can be observed in brain regions particularly sensitive to stress. Telomere length was measured by quantitative-PCR in five brain regions (dorsolateral prefrontal cortex, hippocampus (HIPP), amygdala, nucleus accumbens and substantia nigra (SN)) in major depressive disorder (MDD), bipolar disorder, SZ and normal control subjects (N = 40, 10 subjects per group). We observed significant differences in telomere length across brain regions suggesting variable levels of cell aging, with SN and HIPP having the longest telomeres and the dorsolateral prefrontal cortex the shortest. A significant decrease (P < 0.02) in telomere length was observed specifically in the HIPP of MDD subjects even after controlling for age. In the HIPP of MDD subjects, several genes involved in neuroprotection and in stress response (FKBP5, CRH) showed altered levels of mRNA. Our results suggest the presence of hippocampal stress-mediated accelerated cellular aging in depression. Further studies are needed to investigate the cellular specificity of these findings.

Ultrasound-induced inertial cavitation from gas-stabilizing nanoparticles.

The understanding of cavitation from nanoparticles has been hindered by the inability to control nanobubble size. We present a method to manufacture nanoparticles with a tunable single hemispherical depression (nanocups) of mean diameter 90, 260, or 650 nm entrapping a nanobubble. A modified Rayleigh-Plesset crevice model predicts the inertial cavitation threshold as a function of cavity size and frequency, and is verified experimentally. The ability to tune cavitation nanonuclei and predict their behavior will be useful for applications ranging from cancer therapy to ultrasonic cleaning.