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.

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.

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.

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.

A short report of satisfaction levels amongst Irish trainees in psychiatry with out of hours and emergency assessments.

OBJECTIVES: On-call and crisis psychiatry is a very challenging aspect of psychiatric training. This study aimed to describe the experiences of psychiatric trainees on-call in hospitals, emergency departments and psychiatric units in Ireland. METHODS: In total, 193 psychiatric trainees in Ireland were emailed a survey in 2017. The survey included questions regarding the duties expected of the trainee, frequency of on-call obligations, un-rostered hours worked, level of senior support, assessment facilities available and doctors' satisfaction with the on-call experience. RESULTS: Overall, 68 trainees responded to the survey. In total, 35% of respondents reported dissatisfaction with their experience of on-call and crisis psychiatry, 46% reported that they were not provided with training in risk assessment and 21% of respondents stated that there was not a suitable room available to perform their assessments. CONCLUSIONS: This survey has raised important issues facing those on the frontline of psychiatric services in Ireland. Of particular concern are resource issues faced by trainees and the need for further training and support related to risk assessment when on-call. Remedying these issues may lead to a decreased rate of dropout as well as a safer and better environment for patients and doctors alike.

First demonstration of a fiber optic bolometer on a tokamak plasma (invited).

A fiber optic bolometer (FOB) was demonstrated observing a fusion plasma for the first time at the DIII-D tokamak. A FOB uses a fiber optics-based interferometric technique that is designed to have a high sensitivity to temperature changes [75 mK/(W/m2) responsivity in high vacuum with 0.38 mK noise level] with a negligible susceptibility to electromagnetic interference (EMI) that can be problematic for resistive bolometers in a tokamak environment. A single-channel test apparatus was installed on DIII-D consisting of a measurement FOB and shielded reference FOB. The single-channel FOB showed a negligible increase in the noise level during typical plasma operations (0.39 mK) compared to the benchtop results (0.38 mK), confirming an insignificant EMI impact to the FOB. Comparisons to DIII-D resistive bolometers showed good agreement with the single-channel FOB, indicating that the FOB is comparable to a resistive bolometer when the impulse calibration is applied. The noise-equivalent power density of the calibrated FOB during a plasma operation was 0.55 W/m2 with an average sampling time of 20 ms. The major potential effect of ionizing radiation on the FOB would be the radiation-induced attenuation, which can be efficiently compensated for by adjusting the probing light power.

Measurements of multiple heat flux components at the divertor target by using surface eroding thermocouples (invited).

The Surface Eroding Thermocouple (SETC) is a robust diagnostic utilized in DIII-D to provide fast, edge-localized modes (ELMs) resolved heat flux measurements, in particular in geometric regions that are too shadowed for traditional infrared thermography. In order to further investigate the power dissipation in the divertor region, a combination of flush-mounted and recessed SETCs was developed to assess the effect on surface heating from non-charged particles at the divertor target. Utilizing the Divertor Materials Evaluation System sample exposure platform, the first demonstration of the feasibility of using this new method to distinguish between the heat flux from charged particles and that from neutrals and radiative heating was achieved. This paper details the process of using the combination of flush SETCs and recessed SETCs to measure the multiple heat flux components at the divertor target and further discusses how to determine two important ratios, α (ratio of heat flux from charged particles deposit on recessed SETC to that deposit on flush SETC) and ß (ratio of heat flux from non-charged particles deposit on recessed SETC to that deposit on flush SETC), in the estimation of the heat flux from non-charged particle sources. Using a time dependent ratio α, it was found that ∼50% of the total incident heat flux is attributable to the non-charged particles in the fully detached open divertor in DIII-D. Finally, the new application of similar SETC diagnostics in the Small Angle Slot divertor with a V-like configuration and partial tungsten coated surface (SAS-VW) is also introduced.

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.

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.

Neuronal Growth and Formation of Neuron Networks on Directional Surfaces.

The formation of neuron networks is a process of fundamental importance for understanding the development of the nervous system and for creating biomimetic devices for tissue engineering and neural repair. The basic process that controls the network formation is the growth of an axon from the cell body and its extension towards target neurons. Axonal growth is directed by environmental stimuli that include intercellular interactions, biochemical cues, and the mechanical and geometrical properties of the growth substrate. Despite significant recent progress, the steering of the growing axon remains poorly understood. In this paper, we develop a model of axonal motility, which incorporates substrate-geometry sensing. We combine experimental data with theoretical analysis to measure the parameters that describe axonal growth on micropatterned surfaces: diffusion (cell motility) coefficients, speed and angular distributions, and cell-substrate interactions. Experiments performed on neurons treated with inhibitors for microtubules (Taxol) and actin filaments (Y-27632) indicate that cytoskeletal dynamics play a critical role in the steering mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which geometrical patterns impart high traction forces to the growth cone. These results have important implications for bioengineering novel substrates to guide neuronal growth and promote nerve repair.

Delivering Care to High-Cost High-Need Patients: Lessons Learned in the Development of a Complex Care Primary Care Team.

As part of a population health-focused primary care transformation, in 2019 a health system in Minnesota developed a primary care team to exclusively care for high-cost high-need patients. Through its development and implementation, the team has discovered several key lessons in delivering care to complex patients. These lessons include the benefits of more integrative team-based care, the need and advantages of designated complex care team members, the importance of teamwork both within and outside of the complex care team, the need for frequent communication, and the importance of identifying mental health needs. In addition, there are several areas that require ongoing research and exploration, such as determining when a patient is able to graduate out of the program, how to enhance access to the complex care team, determining appropriate visit characteristics, and model feasibility. While addressing the needs of high cost high need patients is essential to improving quality of care and decreasing health care costs, there are several unique challenges and opportunities that come with caring for this patient population. Although this highly integrated model of care continues to evolve, the initial lessons learned may inform other health systems and care teams undertaking the care of complex patients.

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.

Effects of underfeeding and oral vancomycin on gut microbiome and nutrient absorption in humans.

Direct evidence in humans for the impact of the microbiome on nutrient absorption is lacking. We conducted an extended inpatient study using two interventions that we hypothesized would alter the gut microbiome and nutrient absorption. In each, stool calorie loss, a direct proxy of nutrient absorption, was measured. The first phase was a randomized cross-over dietary intervention in which all participants underwent in random order 3 d of over- and underfeeding. The second was a randomized, double-blind, placebo-controlled pharmacologic intervention using oral vancomycin or matching placebo (NCT02037295). Twenty-seven volunteers (17 men and 10 women, age 35.1 ± 7.3, BMI 32.3 ± 8.0), who were healthy other than having impaired glucose tolerance and obesity, were enrolled and 25 completed the entire trial. The primary endpoints were the effects of dietary and pharmacological intervention on stool calorie loss. We hypothesized that stool calories expressed as percentage of caloric intake would increase with underfeeding compared with overfeeding and increase during oral vancomycin treatment. Both primary endpoints were met. Greater stool calorie loss was observed during underfeeding relative to overfeeding and during vancomycin treatment compared with placebo. Key secondary endpoints were to evaluate the changes in gut microbial community structure as evidenced by amplicon sequencing and metagenomics. We observed only a modest perturbation of gut microbial community structure with under- versus overfeeding but a more widespread change in community structure with reduced diversity with oral vancomycin. Increase in Akkermansia muciniphila was common to both interventions that resulted in greater stool calorie loss. These results indicate that nutrient absorption is sensitive to environmental perturbations and support the translational relevance of preclinical models demonstrating a possible causal role for the gut microbiome in dietary energy harvest.

A simple vacuum suitcase for enabling plasma facing component characterization in fusion devices.

We have demonstrated a vacuum suitcase to transport samples in vacuo to a surface analysis station for characterization of tokamak plasma facing components (PFCs). This technique enables surface analysis at powerful, dedicated stations that are not encumbered by design constraints imposed on them by a tokamak. The vacuum suitcase is an alternative solution to characterizing PFCs using diagnostics that are designed and built around a tokamak. The vacuum suitcase, called the Sample Exposure Probe (SEP), features mobile ultra-high vacuum pumping. Active pumping under high vacuum enables sample transfer between the Lithium Tokamak eXperiment-ß (LTX-ß) and a high resolution X-ray Photoelectron Spectroscopy (XPS) system that is situated close by. A thermocouple inserted in the back of the sample head measures heat flux from the plasma during exposure, and together with a button heater, allows the sample to match the LTX-ß PFCs in high temperature operations. As vacuum conditions are better during transfer and analysis than in the tokamak, less contamination is introduced to the samples. XPS scans on a dedicated analysis station enable peak identification due to higher resolution and signal to noise ratio. A similar probe could be implemented for other fusion devices. The SEP is the first vacuum suitcase implementation for fusion applications that incorporates active pumping.