The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics.

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

The neuroprotective N-terminal amyloid-ß core hexapeptide reverses reactive gliosis and gliotoxicity in Alzheimer's disease pathology models.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by accumulation of extracellular amyloid beta (Aß) and intracellular neurofibrillary tangles, leading to chronic activation of astrocytes and microglia and persistent neuroinflammation. Aß-linked activation of microglia and astrocytes leads to increased intracellular calcium and production of proinflammatory cytokines, impacting the progression of neurodegeneration. An N-terminal Aß fragment (Aß1-15) and a shorter hexapeptide core sequence within the N-Aß fragment (N-Aßcore: Aß10-15) have previously been shown to protect against Aß-induced mitochondrial dysfunction, oxidative stress and apoptosis in neurons and rescue synaptic and spatial memory deficits in an APP/PSEN1 mouse model. Here, we hypothesized that the N-Aß fragment and N-Aßcore are protective against Aß-induced gliotoxicity, promoting a neuroprotective environment and potentially alleviating the characteristically persistent neuroinflammation present in AD. METHODS: We treated ex vivo organotypic brain slice cultures from an aged familial AD mouse model, 5xFAD, with the N-Aßcore and used immunocytochemistry to assess the impact on astrogliosis and microgliosis and alterations in synaptophysin-positive puncta engulfed by microglia. Isolated neuron/glia cultures, mixed glial cultures or a microglial cell line were treated with oligomeric human Aß at concentrations mimicking the pathogenic concentrations (µM) observed in AD in the absence or presence of the non-toxic N-terminal Aß fragments. Resultant changes in synaptic density, gliosis, oxidative stress, mitochondrial dysfunction, apoptosis, and the expression and release of proinflammatory markers were then determined. RESULTS: We demonstrate that the N-terminal Aß fragments mitigated the phenotypic switch leading to astrogliosis and microgliosis induced by pathological concentrations of Aß in mixed glial cultures and organotypic brain slice cultures from the transgenic 5xFAD mouse model, while protecting against Aß-induced oxidative stress, mitochondrial dysfunction and apoptosis in isolated astrocytes and microglia. Moreover, the addition of the N-Aßcore attenuated the expression and release of proinflammatory mediators in microglial cells activated by Aß and rescued microglia-mediated loss of synaptic elements induced by pathological levels of Aß. CONCLUSIONS: Together, these findings indicate the protective functions of the N-terminal Aß fragments extend to reactive gliosis and gliotoxicity induced by Aß, by preventing or reversing glial reactive states indicative of neuroinflammation and synaptic loss central to AD pathogenesis.

Feedback-controlled dynamics of neuronal cells on directional surfaces.

The formation of neuronal networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body and axonal navigation toward target neurons. Axonal growth is guided by the interactions between the tip of the axon (growth cone) and its extracellular environmental cues, which include intercellular interactions, the biochemical landscape around the neuron, and the mechanical and geometrical features of the growth substrate. Here, we present a comprehensive experimental and theoretical analysis of axonal growth for neurons cultured on micropatterned polydimethylsiloxane (PDMS) surfaces. We demonstrate that closed-loop feedback is an essential component of axonal dynamics on these surfaces: the growth cone continuously measures environmental cues and adjusts its motion in response to external geometrical features. We show that this model captures all the characteristics of axonal dynamics on PDMS surfaces for both untreated and chemically modified neurons. We combine experimental data with theoretical analysis to measure key parameters that describe axonal dynamics: diffusion (cell motility) coefficients, speed and angular distributions, and cell-substrate interactions. The experiments performed on neurons treated with Taxol (inhibitor of microtubule dynamics) and Y-27632 (disruptor of actin filaments) indicate that the internal dynamics of microtubules and actin filaments plays a critical role for the proper function of the feedback mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrate to guide neuronal growth and promote nerve repair.

Bi-national outbreak of Salmonella Newport infections linked to onions: the United States experience.

From 2016-2019, dry bulb onions were the suspected cause of three multistate outbreaks in the United States. We investigated a large multistate outbreak of Salmonella Newport infections that caused illnesses in both the United States and Canada in 2020. Epidemiologic, laboratory and traceback investigations were conducted to determine the source of the infections, and data were shared among U.S. and Canadian public health officials. We identified 1127 U.S. illnesses from 48 states with illness onset dates ranging from 19 June to 11 September 2020. Sixty-six per cent of ill people reported consuming red onions in the week before illness onset. Thirty-five illness sub-clusters were identified during the investigation and seventy-four per cent of sub-clusters served red onions to customers during the exposure period. Traceback for the source of onions in illness sub-clusters identified a common onion grower in Bakersfield, CA as the source of red onions, and onions were recalled at this time. Although other strains of Salmonella Newport were identified in environmental samples collected at the Bakersfield, CA grower, extensive environmental and product testing did not yield the outbreak strain. This was the third largest U.S. foodborne Salmonella outbreak in the last 30 years. It is the first U.S. multistate outbreak with a confirmed link to dry bulb onions, and it was nearly 10-fold larger than prior outbreaks with a suspected link to onions. This outbreak is notable for its size and scope, as well as the international data sharing that led to implication of red onions as the primary cause of the outbreak. Although an environmental assessment at the grower identified several factors that likely contributed to the outbreak, no main reason was identified. The expedient identification of the outbreak vehicle and response of multiple public health agencies allowed for recall and removal of product from the marketplace, and rapid messaging to both the public and industry on actions to protect consumers; these features contributed to a decrease in cases and expeditious conclusion of the outbreak.

11C-methionine PET aids localization of microprolactinomas in patients with intolerance or resistance to dopamine agonist therapy.

PURPOSE: To assess the potential for 11C-methionine PET (Met-PET) coregistered with volumetric magnetic resonance imaging (Met-PET/MRCR) to inform clinical decision making in patients with poorly visualized or occult microprolactinomas and dopamine agonist intolerance or resistance. PATIENTS AND METHODS: Thirteen patients with pituitary microprolactinomas, and who were intolerant (n = 11) or resistant (n = 2) to dopamine agonist therapy, were referred to our specialist pituitary centre for Met-PET/MRCR between 2016 and 2020. All patients had persistent hyperprolactinemia and were being considered for surgical intervention, but standard clinical MRI had shown either no visible adenoma or equivocal appearances. RESULTS: In all 13 patients Met-PET/MRCR demonstrated a single focus of avid tracer uptake. This was localized either to the right or left side of the sella in 12 subjects. In one patient, who had previously undergone surgery for a left-sided adenoma, recurrent tumor was unexpectedly identified in the left cavernous sinus. Five patients underwent endoscopic transsphenoidal selective adenomectomy, with subsequent complete remission of hyperprolactinaemia and normalization of other pituitary function; three patients are awaiting surgery. In the patient with inoperable cavernous sinus disease PET-guided stereotactic radiosurgery (SRS) was performed with subsequent near-normalization of serum prolactin. Two patients elected for a further trial of medical therapy, while two declined surgery or radiotherapy and chose to remain off medical treatment. CONCLUSIONS: In patients with dopamine agonist intolerance or resistance, and indeterminate pituitary MRI, molecular (functional) imaging with Met-PET/MRCR can allow precise localization of a microprolactinoma to facilitate selective surgical adenomectomy or SRS.

Covid-19 and the Impact on Referrals to Psychiatry in those 65 years and Older.

Aim To measure the impact of Covid-19 on the mental health of those 65 years and older, referrals to psychiatry in this Emergency Department (ED) were examined. This was likely the 'tip of the iceberg' in difficulties in this cohort and may predict patterns in a future 'tsunami' of cases. Methods A review of referrals from ED in those ≥ 65 years was conducted, from April to September in 2019 and 2020. Number of presentations, referral reason, alcohol issues, presentation method and assessment outcome were examined. Results From May 2020, there was increased referrals in all ages, except those aged 65 and older. Only 6.7% of referrals were ≥ 65years in 2020 (11% in 2019), with more referred for anxiety, suicidality and overdose, with no BPSD (behavioural and psychological symptoms of dementia) referrals recorded. There was an increase presenting with psychosis secondary to mental illness, alcohol issues and brought by emergency services, with a decrease in those linked with services. Conclusions There was a probable unmet burden of psychiatric needs in this age-group with potentially increased distress and reduced supports, in less presentations. Difficulties providing services during this period and lack of presentations such as BPSD, raises concerns for older patients and a future 'tsunami' of presentations.

Exit strategies: optimising feasible surveillance for detection, elimination, and ongoing prevention of COVID-19 community transmission.

BACKGROUND: Following implementation of strong containment measures, several countries and regions have low detectable community transmission of COVID-19. We developed an efficient, rapid, and scalable surveillance strategy to detect remaining COVID-19 community cases through exhaustive identification of every active transmission chain. We identified measures to enable early detection and effective management of any reintroduction of transmission once containment measures are lifted to ensure strong containment measures do not require reinstatement. METHODS: We compared efficiency and sensitivity to detect community transmission chains through testing of the following: hospital cases; fever, cough and/or ARI testing at community/primary care; and asymptomatic testing; using surveillance evaluation methods and mathematical modelling, varying testing capacities, reproductive number (R) and weekly cumulative incidence of COVID-19 and non-COVID-19 respiratory symptoms using data from Australia. We assessed system requirements to identify all transmission chains and follow up all cases and primary contacts within each chain, per million population. RESULTS: Assuming 20% of cases are asymptomatic and 30% of symptomatic COVID-19 cases present for testing, with R = 2.2, a median of 14 unrecognised community cases (8 infectious) occur when a transmission chain is identified through hospital surveillance versus 7 unrecognised cases (4 infectious) through community-based surveillance. The 7 unrecognised community upstream cases are estimated to generate a further 55-77 primary contacts requiring follow-up. The unrecognised community cases rise to 10 if 50% of cases are asymptomatic. Screening asymptomatic community members cannot exhaustively identify all cases under any of the scenarios assessed. The most important determinant of testing requirements for symptomatic screening is levels of non-COVID-19 respiratory illness. If 4% of the community have respiratory symptoms, and 1% of those with symptoms have COVID-19, exhaustive symptomatic screening requires approximately 11,600 tests/million population using 1/4 pooling, with 98% of cases detected (2% missed), given 99.9% sensitivity. Even with a drop in sensitivity to 70%, pooling was more effective at detecting cases than individual testing under all scenarios examined. CONCLUSIONS: Screening all acute respiratory disease in the community, in combination with exhaustive and meticulous case and contact identification and management, enables appropriate early detection and elimination of COVID-19 community transmission. An important component is identification, testing, and management of all contacts, including upstream contacts (i.e. potential sources of infection for identified cases, and their related transmission chains). Pooling allows increased case detection when testing capacity is limited, even given reduced test sensitivity. Critical to the effectiveness of all aspects of surveillance is appropriate community engagement, messaging to optimise testing uptake and compliance with other measures.

Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics.

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common genetic cause of autosomal and sporadic Parkinson's disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation-all of which could lead to neurodegeneration associated with PD.

Improvements in Clinical Outcomes in Children with Cystic Fibrosis aged Six and 16 years.

Aims Our aim was to assess if outcomes for cystic fibrosis (CF) patients at six & sixteen years of age have improved in the last 17 years looking at FEV1, BMI and death. Methods A retrospective observational study using a prospectively maintained database of CF patients at Cork University Hospital. Results 84 patients were included in the 16-year-old data and 89 patients were included in the six-year-old data. The mean FEV1 and BMI (16 years) for the 2002-2007 group was 72.9±21.0% and 18.9±2.53 respectively, 2008-2013 group was 75.4±27.2% and 19.8±2.7 and for the 2014-2018 group was 95.2±16.0% and 22.9±4.1. The percentage of patients (16 years) with chronic pseudomonas status was 37.9% (11/30) in the 2002-2007 group, 51.6 % (16/31) in the 2008-2013 group and 4.2% (1/24) in the 2014-2018 group. The relationship between FEV1 and FVC with BMI remained significant in multivariate analysis (P <0.001). The mean FEV1 (six years) for the 2002-2007 group was 90.7±16.1%, 2008-2013 group was 99.3±17.9% and for the 2014-2018 group was 100.9±15.8%. Conclusions Improvements in FEV1 and BMI aged six and 16 years are notable as well as a significant decline in the number of patients with chronic pseudomonas.

Admission cardiotocography versus intermittent auscultation of the fetal heart in low-risk pregnancy during evaluation for possible labour admission - a multicentre randomised trial: the ADCAR trial.

OBJECTIVE: To assess the effect of admission cardiotocography (ACTG) versus intermittent auscultation (IA) of the fetal heart (FH) in low-risk pregnancy during assessment for possible labour on caesarean section rates. DESIGN: A parallel multicentre randomised trial. SETTING: Three maternity units in the Republic of Ireland. POPULATION: Healthy, low-risk pregnant women, at term and ≥ 18 years old, who provided written informed consent. METHODS: Women were randomised to receive IA of the FH or 20 minutes ACTG on admission for possible labour onset, using remote telephone randomisation. Both groups received IA during labour, with conversion to continuous CTG as clinically indicated. MAIN OUTCOME MEASURES: Caesarean section (primary outcome), obstetric interventions (e.g. continuous CTG during labour, fetal blood sampling, augmentation of labour) and neonatal morbidity (e.g. metabolic acidosis, admission to the neonatal intensive care unit, neonatal death). RESULTS: Based on 3034 women (1513 and 1521 randomised to IA and ACTG, respectively), there was no statistical difference between the groups in caesarean section [130 (8.6%) and 105 (6.9%) for IA and ACTG groups, respectively; relative risk (RR) 1.24; 95% CI 0.97-1.58], or in any other outcome except for use of continuous CTG during labour, which was lower in the IA group (RR 0.90, 95% CI 0.86-0.93). CONCLUSION: Our study demonstrates no differences in obstetric or neonatal outcomes between IA and ACTG for women with possible labour onset, other than an increased risk for continuous CTG in women receiving ACTG. TWEETABLE ABSTRACT: No differences in outcomes between intermittent auscultation and admission cardiotocography for women with possible labour onset.

Meconium Ileus in Two Irish Newborns: The Presenting Feature of Cystic Fibrosis

Introduction Meconium Ileus (MI) is the presenting feature of CF in approximately 10-15% of cases. This report outlines the clinical presentation, imaging and management of two neonates with MI and subsequent diagnosis of Cystic Fibrosis (CF). Methods A retrospective chart review was performed to evaluate the clinical course of two neonates with MI. Results Case 1 and 2 presented clinically with signs of abdominal obstruction. Subsequent laparotomies confirmed MI. MI is strongly associated with CF and CF is the most common genetically inherited disease in Ireland. Genetic testing was positive for a homozygous ∆ F508 mutation in both case 1 and 2, securing a diagnosis of MI secondary to CF. Conclusion Our cases highlight that all infants born in Ireland with MI should be considered as CF positive until proven otherwise.

Evaluation of a lysostaphin-fusion protein as a dry-cow therapy for Staphylococcus aureus mastitis in dairy cattle.

This study evaluated the efficacy of a recombinant lysostaphin fused to a protein transduction domain (rLYS-PTD) as a dry-cow therapy for the treatment of experimentally induced chronic, subclinical Staphylococcus aureus mastitis. Twenty-two Holstein dairy cows were experimentally infected with Staph. aureus in a single pair of diagonal mammary quarters approximately 45d before dry off. Staphylococcus aureus-infected mammary quarters of cows were randomly assigned to 1 of 2 treatment groups at dry off: (1) 279mg of rLYS-PTD in 50mL of vehicle (n=11 cows; 22 quarters) or (2) 50mL of vehicle solution (n=11 cows; 22 quarters) by intramammary infusion. All cows were followed for 30d postpartum to determine cure rates using bacteriologic culture, somatic cell counts, and clinical mastitis scores. No cures were recorded in either the treatment or control groups. Milk somatic cell count, bacterial colony counts, and mastitis scores did not significantly differ between treatment groups. In conclusion, rLYS-PTD was not an effective dry-cow therapeutic for chronic, subclinical Staph. aureus mastitis at the tested dose and formulation.

Irish Medical Students Understanding of the Intern Year.

Upon completion of medical school in Ireland, graduates must make the transition to becoming interns. The transition into the intern year may be described as challenging as graduates assume clinical responsibilities. Historically, a survey of interns in 1996 found that 91% felt unprepared for their role. However, recent surveys in 2012 have demonstrated that this is changing with preparedness rates reaching 52%. This can be partially explained by multiple initiatives at the local and national level. Our study aimed evaluate medical student understanding of the intern year and associated factors. An online, cross-sectional survey was sent out to all Irish medical students in 2013 and included questions regarding their understanding of the intern year. Two thousand, two hundred and forty-eight students responded, with 1,224 (55.4%) of students agreeing or strongly agreeing that they had a good understanding of what the intern year entails. This rose to 485 (73.7%) among senior medical students. Of junior medical students, 260 (42.8%) indicated they understood what the intern year, compared to 479 (48.7%) of intermediate medical students. Initiatives to continue improving preparedness for the intern year are essential in ensuring a smooth and less stressful transition into the medical workforce.

Novel data interpretation method for DIII-D divertor retarding field energy analyzer with 3-D particle-in-cell simulations.

A novel data interpretation process that utilizes comprehensive particle-in-cell (PIC) simulations is developed for the new retarding field energy analyzer (RFEA) currently being constructed at DIII-D for the lower divertor using the Divertor Material Evaluation System. This probe is expected to survive a heat load of up to 100 MW/m2 for up to 5 s and reliably measure the main ion temperature (Ti) on the divertor target ranging from 10 to 200 eV. These extreme conditions posed significant engineering limitations on the probe geometry, thus extensive validation work has been performed. The conventional fitting method for the RFEA I-V characteristics is based on a simplified 1-D model without considering the ion space charge inside the probe cavity and may not be sufficient for probes designed for the DIII-D divertor environment. In this article, a more realistic description of the particle propagation process within the RFEA cavity is achieved by including both 3-D geometric effects and ion space charge in the PIC simulations, and the capability to reconstruct the ion energy distribution functions is demonstrated with reasonable consistency.

Molecular basis of proton-sensing by G protein-coupled receptors.

Three proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement of putative proton sensing residues in the active state. With a newly developed deep mutational scanning approach, we determined the functional importance of every residue in proton activation for GPR68 by generating ~9,500 mutants and measuring effects on signaling and surface expression. This unbiased screen revealed that, unlike other proton-sensitive cell surface channels and receptors, no single site is critical for proton recognition in GPR68. Instead, a network of titratable residues extend from the extracellular surface to the transmembrane region and converge on canonical class A GPCR activation motifs to activate proton-sensing GPCRs. More broadly, our approach integrating structure and unbiased functional interrogation defines a new framework for understanding the rich complexity of GPCR signaling.

Tetrathiomolybdate-associated copper depletion decreases circulating endothelial progenitor cells in women with breast cancer at high risk of relapse.

BACKGROUND: Bone marrow-derived endothelial progenitor cells (EPCs) are critical for metastatic progression. This study explores the effect of tetrathiomolybdate (TM), an anti-angiogenic copper chelator, on EPCs in patients at high risk for breast cancer recurrence. PATIENTS AND METHODS: This phase 2 study enrolled breast cancer patients with stage 3 and stage 4 without evidence of disease (NED), and stage 2 if triple-negative. TM 100 mg orally was administered to maintain ceruloplasmin <17 mg/dl for 2 years or until relapse. The primary end point was change in EPCs. RESULTS: Forty patients (28 stage 2/3, 12 stage 4 NED) were enrolled. Seventy-five percent patients achieved the copper depletion target by 1 month. Ninety-one percent of triple-negative patients copper-depleted compared with 41% luminal subtypes. In copper-depleted patients only, there was a significant reduction in EPCs/ml by 27 (P = 0.04). Six patients relapsed while on study, of which only one patient had EPCs maintained below baseline. The 10-month relapse-free survival was 85.0% (95% CI 74.6%-96.8%). Only grade 3/4 toxicity was hematologic: neutropenia (3.1% of cycles), febrile neutropenia (0.2%), and anemia (0.2%). CONCLUSIONS: TM is safe and appears to maintain EPCs below baseline in copper-depleted patients. TM may promote tumor dormancy and ultimately prevent relapse.

High sensitivity of Aeolus UV surface returns to surface reflectivity.

Global ultraviolet (UV) surface reflectivity climatologies are all composed from daylight passive remote sensing observations of reflected UV light, generally integrated over a distribution of attainable reflection directions. We discovered the sensitivity of Aeolus lidar surface returns (LSR) to surface characteristics, providing the first evidence that active remote sensing can be effectively used for retrieving unidirectional UV surface reflectivity on global scales. LSR reproduces surface reflectivity monthly changes in Sahara, which are visible in the Lambertian Equivalent Reflectivity (LER) climatologies from TROPOMI and GOME-2. Very high correlations (r > 0.90) between gridded LSR and the LER climatologies are reported at global and regional scales for 36 different regions. Three clear land cover gradients are discerned from Aeolus LSR signal: (1) water/land, (2) vegetation/arid areas and (3) no snow/snow. The strongest LSR signal was retrieved over snow, while over vegetation, we found moderate negative agreement (r < - 0.60) between LSR and vegetation index proxy. Overall, the success of the first active remote sensing method for retrieving unidirectional UV surface reflectivity using Aeolus is demonstrated. Our approach can be effectively used to detect unresolved land and, especially, snow cover changes in high latitudes because, unlike passive instruments, Aeolus also provided nighttime observations.