The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3.

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (Dnm1-3), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

Three-year follow-up of suicide prevention-focused group therapy for veterans.

The purpose of this study was to determine the long-term effects of a suicide prevention-focused group therapy for veterans recently discharged from an inpatient psychiatry setting following a suicidal crisis. There was interest in examining the impact of mechanisms of change identified in previous research on the group, including group cohesion, working alliance, and group sessions attended. Data were abstracted from the electronic medical record 3 years following completion of a previous study that involved the group therapy. A series of generalized linear and logistic mixed models were conducted to measure the associations between group cohesion, working alliance, session attendance, and health service utilization and suicide attempts. Thirty randomly selected veterans from the original sample completed a semistructured interview to discuss their experience in the group therapy. Study team members reviewed each transcription to identify themes related to veterans' experiences in the suicide prevention-focused group therapy. No suicides were observed in the 3-year follow-up period. When examining the full sample (N = 134), session attendance and inpatient hospitalization were not significantly associated but were positively associated after removing subjects who attended zero sessions (N = 93). Higher group cohesion was associated with a reduced likelihood of inpatient psychiatric hospitalization and greater engagement in outpatient mental health services. Four themes emerged regarding veterans' experience in the group through an analysis of the semistructured interviews. Suicide prevention-focused group therapy among veteran service members was not associated with an elevated risk of mortality. Future research is needed to further elucidate mechanisms of change and moderators of response. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Molecular identification of wide-field amacrine cells in mouse retina that encode stimulus orientation.

Visual information processing is sculpted by a diverse group of inhibitory interneurons in the retina called amacrine cells. Yet, for most of the >60 amacrine cell types, molecular identities and specialized functional attributes remain elusive. Here, we developed an intersectional genetic strategy to target a group of wide-field amacrine cells (WACs) in mouse retina that co-express the transcription factor Bhlhe22 and the Kappa Opioid Receptor (KOR; B/K WACs). B/K WACs feature straight, unbranched dendrites spanning over 0.5 mm (∼15° visual angle) and produce non-spiking responses to either light increments or decrements. Two-photon dendritic population imaging reveals Ca 2+ signals tuned to the physical orientations of B/K WAC dendrites, signifying a robust structure-function alignment. B/K WACs establish divergent connections with multiple retinal neurons, including unexpected connections with non-orientation-tuned ganglion cells and bipolar cells. Our work sets the stage for future comprehensive investigations of the most enigmatic group of retinal neurons: WACs.

Multiple Calcium Channel Types with Unique Expression Patterns Mediate Retinal Signaling at Bipolar Cell Ribbon Synapses.

Retinal bipolar cells (BCs) compose the canonical vertical excitatory pathway that conveys photoreceptor output to inner retinal neurons. Although synaptic transmission from BC terminals is thought to rely almost exclusively on Ca2+ influx through voltage-gated Ca2+ (CaV) channels mediating L-type currents, the molecular identity of CaV channels in BCs is uncertain. Therefore, we combined molecular and functional analyses to determine the expression profiles of CaV α1, ß, and α2δ subunits in mouse rod bipolar (RB) cells, BCs from which the dynamics of synaptic transmission are relatively well-characterized. We found significant heterogeneity in CaV subunit expression within the RB population from mice of either sex, and significantly, we discovered that transmission from RB synapses was mediated by Ca2+ influx through P/Q-type (CaV2.1) and N-type (CaV2.2) conductances as well as the previously-described L-type (CaV1) and T-type (CaV3) conductances. Furthermore, we found both CaV1.3 and CaV1.4 proteins located near presynaptic ribbon-type active zones in RB axon terminals, indicating that the L-type conductance is mediated by multiple CaV1 subtypes. Similarly, CaV3 α1, ß, and α2δ subunits also appear to obey a "multisubtype" rule, i.e., we observed a combination of multiple subtypes, rather than a single subtype as previously thought, for each CaV subunit in individual cells.SIGNIFICANCE STATEMENT Bipolar cells (BCs) transmit photoreceptor output to inner retinal neurons. Although synaptic transmission from BC terminals is thought to rely almost exclusively on Ca2+ influx through L-type voltage-gated Ca2+ (CaV) channels, the molecular identity of CaV channels in BCs is uncertain. Here, we report unexpectedly high molecular diversity of CaV subunits in BCs. Transmission from rod bipolar (RB) cell synapses can be mediated by Ca2+ influx through P/Q-type (CaV2.1) and N-type (CaV2.2) conductances as well as the previously-described L-type (CaV1) and T-type (CaV3) conductances. Furthermore, CaV1, CaV3, ß, and α2δ subunits appear to obey a "multisubtype" rule, i.e., a combination of multiple subtypes for each subunit in individual cells, rather than a single subtype as previously thought.

Making genomic surveillance deliver: A lineage classification and nomenclature system to inform rabies elimination.

The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus, however the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 96 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, as well as areas of persistence and routes of virus movement, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as countries work towards eliminating dog-mediated rabies, as well as having potential for broader application to the surveillance of other viruses.

The evolutionary dynamics and epidemiological history of hepatitis C virus genotype 6, including unique strains from the Li community of Hainan Island, China.

Hepatitis C virus (HCV) is a highly diverse pathogen that frequently establishes a chronic long-term infection, but the origins and drivers of HCV diversity in the human population remain unclear. Previously unidentified strains of HCV genotype 6 (gt6) were recently discovered in chronically infected individuals of the Li ethnic group living in Baisha County, Hainan Island, China. The Li community, who were early settlers on Hainan Island, has a distinct host genetic background and cultural identity compared to other ethnic groups on the island and mainland China. In this report, we generated 33 whole virus genome sequences to conduct a comprehensive molecular epidemiological analysis of these novel gt6 strains in the context of gt6 isolates present in Southeast Asia. With the exception of one gt6a isolate, the Li gt6 sequences formed three novel clades from two lineages which constituted 3 newly assigned gt6 subtypes and 30 unassigned strains. Using Bayesian inference methods, we dated the most recent common ancestor for all available gt6 whole virus genome sequences to approximately 2767 bce (95 per cent highest posterior density (HPD) intervals, 3670-1397 bce), which is far earlier than previous estimates. The substitution rate was 1.20 × 10-4 substitutions/site/year (s/s/y), and this rate varied across the genome regions, from 1.02 × 10-5 s/s/y in the 5'untranslated region (UTR) region to 3.07 × 10-4 s/s/y in E2. Thus, our study on an isolated ethnic minority group within a small geographical area of Hainan Island has substantially increased the known diversity of HCV gt6, already acknowledged as the most diverse HCV genotype. The extant HCV gt6 sequences from this study were probably transmitted to the Li through at least three independent events dating perhaps from around 4,000 years ago. This analysis describes deeper insight into basic aspects of HCV gt6 molecular evolution including the extensive diversity of gt6 sequences in the isolated Li ethnic group.

Analysis of rod/cone gap junctions from the reconstruction of mouse photoreceptor terminals.

Electrical coupling, mediated by gap junctions, contributes to signal averaging, synchronization, and noise reduction in neuronal circuits. In addition, gap junctions may also provide alternative neuronal pathways. However, because they are small and especially difficult to image, gap junctions are often ignored in large-scale 3D reconstructions. Here, we reconstruct gap junctions between photoreceptors in the mouse retina using serial blockface-scanning electron microscopy, focused ion beam-scanning electron microscopy, and confocal microscopy for the gap junction protein Cx36. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules at sites of Cx36 labeling, with approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. We estimate a mean of 86 Cx36 channels per rod/cone pair, which may provide a maximum conductance of ~1200 pS, if all gap junction channels were open. This is comparable to the maximum conductance previously measured between rod/cone pairs in the presence of a dopamine antagonist to activate Cx36, suggesting that the open probability of gap junction channels can approach 100% under certain conditions.

Neurons can talk to each other in two ways: they can send chemical messengers across specialized junctions between two cells, or they can directly pass electrical signals to one another. This latter process is made possible by gap junctions, a system of channel-like structures which connect neighbouring cells and let ions move between them. In most neurons, gap junction channels are made from a specialized protein called connexin 36. Gap junctions are small, difficult to observe, and therefore often ignored by researchers studying neural circuits. In response, Ishibashi et al. focused on nerve cells in the mouse retina, in particular the cones (which detect color during the day) and the rods (which are essential for night vision). Gap junctions between rods and cones allow them to communicate; for example, they enable rod signals to directly activate cones. This provides an alternative route for rod signaling known as the 'secondary rod pathway', which seems to be open at night and switches to closed around dawn. Both rods and cones only produce connexin 36, so Ishibashi et al. labeled these proteins with fluorescent tags to pinpoint gap junctions. This showed that each cone makes around 50 gap junctions with nearby rods; however, gap junctions were not detected between cells of the same type. In addition, 3D reconstruction helped to establish the length of each gap junction. Further experiments showed that a typical rod was connected to a cone by about 80 connexin 36 channels. Finally, calculations revealed that the gap junction channels would all need to open to account for the level of electrical activity required for the secondary rod pathway. This suggests that gap junctions may be much more active and important than previously thought. The work by Ishibashi et al. provides a new understanding of the number, size and activity of gap junctions in the retina, potentially paving the way to prevent diseases where light-sensing cells degenerate and cause blindness.

Characteristics Associated With Disparities Among Older Adults in Coronavirus Disease 2019 Outcomes in an Academic Health Care System.

BACKGROUND: An improved understanding of the coronavirus disease 2019 (COVID-19) pandemic is needed to identify predictors of outcomes among older adults with COVID-19. OBJECTIVE: The objective of this study was to examine patient and health system factors predictive of in-hospital mortality, intensive care unit (ICU) admission, and readmission among patients with COVID-19. DESIGN, SETTING, AND PARTICIPANTS: A cohort study of patients aged 18 years and older with COVID-19 discharged from 5 New York hospitals within the Mount Sinai Health System (March 1, 2020-June 30, 2020). MEASURES: Patient-level characteristics (age, sex, race/ethnicity, comorbidities/serious illness, transfer from skilled nursing facility, severe acute respiratory syndrome coronavirus 2 viral load, Sequential Organ Failure Assessment score, treatments); hospital characteristics. OUTCOMES: All-cause in-hospital mortality; ICU admission; 30-day readmission. RESULTS: Among 7556 subjects, mean age 61.1 (62.0) years; 1556 (20.6%) died, 949 (12.6%) had an ICU admission, and 227 (9.1%) had a 30-day readmission. Increased age [aged 55-64: odds ratio (OR), 3.28; 95% confidence interval (CI), 2.41-4.46; aged 65-74: OR, 4.67; 95% CI, 3.43-6.35; aged 75-84: OR, 10.73; 95% CI, 7.77-14.81; aged 85 y and older: OR, 20.57; 95% CI, 14.46-29.25] and comorbidities (OR, 1.11; 95% CI, 1.16, 2.13) were independent risk factors for in-hospital mortality. Yet older adults (aged 55-64 y: OR, 0.56; 95% CI, 0.40-0.77; aged 65-74: OR, 0.46; 95% CI, 0.33-0.65; aged 75-84: OR, 0.27; 95% CI, 0.18-0.40; aged above 85 y: OR, 0.21; 95% CI, 0.13-0.34) and those with Medicaid (OR, 0.74; 95% CI, 0.56-0.99) were less likely to be admitted to the ICU. Race/ethnicity, crowding, population density, and health system census were not associated with study outcomes. CONCLUSIONS: Increased age was the single greatest independent risk factor for mortality. Comorbidities and serious illness were independently associated with mortality. Understanding these risk factors can guide medical decision-making for older adults with COVID-19. Older adults and those admitted from a skilled nursing facility were half as likely to be admitted to the ICU. This finding requires further investigation to understand how age and treatment preferences factored into resource allocation.

Dendro-somatic synaptic inputs to ganglion cells contradict receptive field and connectivity conventions in the mammalian retina.

The morphology of retinal neurons strongly influences their physiological function. Ganglion cell (GC) dendrites ramify in distinct strata of the inner plexiform layer (IPL) so that GCs responding to light increments (ON) or decrements (OFF) receive appropriate excitatory inputs. This vertical stratification prescribes response polarity and ensures consistent connectivity between cell types, whereas the lateral extent of GC dendritic arbors typically dictates receptive field (RF) size. Here, we identify circuitry in mouse retina that contradicts these conventions. AII amacrine cells are interneurons understood to mediate "crossover" inhibition by relaying excitatory input from the ON layer to inhibitory outputs in the OFF layer. Ultrastructural and physiological analyses show, however, that some AIIs deliver powerful inhibition to OFF GC somas and proximal dendrites in the ON layer, rendering the inhibitory RFs of these GCs smaller than their dendritic arbors. This OFF pathway, avoiding entirely the OFF region of the IPL, challenges several tenets of retinal circuitry. These results also indicate that subcellular synaptic organization can vary within a single population of neurons according to their proximity to potential postsynaptic targets.

Access to Palliative Care Consultation for Hospitalized Adults with COVID-19 in an Urban Health System: Were There Disparities at the Peak of the Pandemic?

Background: Palliative care (PC) services expanded rapidly to meet the needs of coronavirus disease 2019 (COVID-19) patients, yet little is known about which patients were referred for PC consultation during the pandemic. Objective: Examine factors predictive of PC consultation for COVID-19 patients. Design: Retrospective cohort study of COVID-19 patients discharged from four hospitals (March 1-June 30, 2020). Exposures: Patient demographic, socioeconomic, and clinical factors and hospital-level characteristics. Outcome Measurement: Inpatient PC consultation. Results: Of 4319 hospitalized COVID-19 patients, 581 (14%) received PC consultation. Increasing age, serious illness (cancer, chronic obstructive pulmonary disease, and dementia), greater illness severity, and admission to the quaternary hospital were associated with receipt of PC consultation. There was no association between PC consultation and race/ethnicity, household crowding, insurance status, or hospital-factors, including inpatient, emergency department, and intensive care unit census. Conclusions: Although site variation existed, the highest acuity patients were most likely to receive PC consultation without racial/ethnic or socioeconomic disparities.

Computational and Molecular Properties of Starburst Amacrine Cell Synapses Differ With Postsynaptic Cell Type.

A presynaptic neuron can increase its computational capacity by transmitting functionally distinct signals to each of its postsynaptic cell types. To determine whether such computational specialization occurs over fine spatial scales within a neurite arbor, we investigated computation at output synapses of the starburst amacrine cell (SAC), a critical component of the classical direction-selective (DS) circuit in the retina. The SAC is a non-spiking interneuron that co-releases GABA and acetylcholine and forms closely spaced (<5 µm) inhibitory synapses onto two postsynaptic cell types: DS ganglion cells (DSGCs) and neighboring SACs. During dynamic optogenetic stimulation of SACs in mouse retina, whole-cell recordings of inhibitory postsynaptic currents revealed that GABAergic synapses onto DSGCs exhibit stronger low-pass filtering than those onto neighboring SACs. Computational analyses suggest that this filtering difference can be explained primarily by presynaptic properties, rather than those of the postsynaptic cells per se. Consistent with functionally diverse SAC presynapses, blockade of N-type voltage-gated calcium channels abolished GABAergic currents in SACs but only moderately reduced GABAergic and cholinergic currents in DSGCs. These results jointly demonstrate how specialization of synaptic outputs could enhance parallel processing in a compact interneuron over fine spatial scales. Moreover, the distinct transmission kinetics of GABAergic SAC synapses are poised to support the functional diversity of inhibition within DS circuitry.

Rapid feedback on hospital onset SARS-CoV-2 infections combining epidemiological and sequencing data.

Background: Rapid identification and investigation of healthcare-associated infections (HCAIs) is important for suppression of SARS-CoV-2, but the infection source for hospital onset COVID-19 infections (HOCIs) cannot always be readily identified based only on epidemiological data. Viral sequencing data provides additional information regarding potential transmission clusters, but the low mutation rate of SARS-CoV-2 can make interpretation using standard phylogenetic methods difficult. Methods: We developed a novel statistical method and sequence reporting tool (SRT) that combines epidemiological and sequence data in order to provide a rapid assessment of the probability of HCAI among HOCI cases (defined as first positive test >48 hr following admission) and to identify infections that could plausibly constitute outbreak events. The method is designed for prospective use, but was validated using retrospective datasets from hospitals in Glasgow and Sheffield collected February-May 2020. Results: We analysed data from 326 HOCIs. Among HOCIs with time from admission ≥8 days, the SRT algorithm identified close sequence matches from the same ward for 160/244 (65.6%) and in the remainder 68/84 (81.0%) had at least one similar sequence elsewhere in the hospital, resulting in high estimated probabilities of within-ward and within-hospital transmission. For HOCIs with time from admission 3-7 days, the SRT probability of healthcare acquisition was >0.5 in 33/82 (40.2%). Conclusions: The methodology developed can provide rapid feedback on HOCIs that could be useful for infection prevention and control teams, and warrants further prospective evaluation. The integration of epidemiological and sequence data is important given the low mutation rate of SARS-CoV-2 and its variable incubation period. Funding: COG-UK HOCI funded by COG-UK consortium, supported by funding from UK Research and Innovation, National Institute of Health Research and Wellcome Sanger Institute.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

Atoh7-independent specification of retinal ganglion cell identity.

Retinal ganglion cells (RGCs) relay visual information from the eye to the brain. RGCs are the first cell type generated during retinal neurogenesis. Loss of function of the transcription factor Atoh7, expressed in multipotent early neurogenic retinal progenitors leads to a selective and essentially complete loss of RGCs. Therefore, Atoh7 is considered essential for conferring competence on progenitors to generate RGCs. Despite the importance of Atoh7 in RGC specification, we find that inhibiting apoptosis in Atoh7-deficient mice by loss of function of Bax only modestly reduces RGC numbers. Single-cell RNA sequencing of Atoh7;Bax-deficient retinas shows that RGC differentiation is delayed but that the gene expression profile of RGC precursors is grossly normal. Atoh7;Bax-deficient RGCs eventually mature, fire action potentials, and incorporate into retinal circuitry but exhibit severe axonal guidance defects. This study reveals an essential role for Atoh7 in RGC survival and demonstrates Atoh7-dependent and Atoh7-independent mechanisms for RGC specification.

Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen.

Virus host shifts are generally associated with novel adaptations to exploit the cells of the new host species optimally. Surprisingly, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has apparently required little to no significant adaptation to humans since the start of the Coronavirus Disease 2019 (COVID-19) pandemic and to October 2020. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects evidence for significant positive episodic diversifying selection acting at the base of the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in these ancestral bat hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor about 1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. While an undiscovered "facilitating" intermediate species cannot be discounted, collectively, our results support the progenitor of SARS-CoV-2 being capable of efficient human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans, which created a relatively generalist virus.

Calmodulin Bidirectionally Regulates Evoked and Spontaneous Neurotransmitter Release at Retinal Ribbon Synapses.

For decades, a role for the Ca2+-binding protein calmodulin (CaM) in Ca2+-dependent presynaptic modulation of synaptic transmission has been recognized. Here, we investigated the influence of CaM on evoked and spontaneous neurotransmission at rod bipolar (RB) cell→AII amacrine cell synapses in the mouse retina. Our work was motivated by the observations that expression of CaM in RB axon terminals is extremely high and that [Ca2+] in RB terminals normally rises sufficiently to saturate endogenous buffers, making tonic CaM activation likely. Taking advantage of a model in which RBs can be stimulated by expressed channelrhodopsin-2 (ChR2) to avoid dialysis of the presynaptic terminal, we found that inhibition of CaM dramatically decreased evoked release by inhibition of presynaptic Ca channels while at the same time potentiating both Ca2+-dependent and Ca2+-independent spontaneous release. Remarkably, inhibition of myosin light chain kinase (MLCK), but not other CaM-dependent targets, mimicked the effects of CaM inhibition on evoked and spontaneous release. Importantly, initial antagonism of CaM occluded the effect of subsequent inhibition of MLCK on spontaneous release. We conclude that CaM, by acting through MLCK, bidirectionally regulates evoked and spontaneous release at retinal ribbon synapses.

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:evbc@unj-jena.de.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.