Cellular-resolution gene expression mapping reveals organization in the head ganglia of the gastropod, Berghia stephanieae.

Gastropod molluscs such as Aplysia, Lymnaea, and Tritonia have been important for determining fundamental rules of motor control, learning, and memory because of their large, individually identifiable neurons. Yet only a small number of gastropod neurons have known molecular markers, limiting the ability to establish brain-wide structure-function relations. Here we combine high-throughput, single-cell RNA sequencing with in situ hybridization chain reaction in the nudibranch Berghia stephanieae to identify and visualize the expression of markers for cell types. Broad neuronal classes were characterized by genes associated with neurotransmitters, like acetylcholine, glutamate, serotonin, and GABA, as well as neuropeptides. These classes were subdivided by other genes including transcriptional regulators and unannotated genes. Marker genes expressed by neurons and glia formed discrete, previously unrecognized regions within and between ganglia. This study provides the foundation for understanding the fundamental cellular organization of gastropod nervous systems.

Egg-laying hormone expression in identified neurons across developmental stages and reproductive states of the nudibranch Berghia stephanieae.

Neuropeptides play essential roles in coordinating reproduction. Egg-laying hormone (ELH) is conserved in genetic sequence and behavioral function across molluscs, where neuronal clusters secrete ELH to modulate and induce egg-laying. Here we investigated ELH in the nudibranch mollusc, Berghia stephanieae. ELH preprohormone gene orthologs, which showed clade-specific differences at the C-terminus of the predicted bioactive peptide, were identified in brain transcriptomes across several nudipleuran species, including B. stephanieae. ELH shares deep homology with the corticotropin-releasing hormone gene family, which has roles broadly in stress response. Injection of synthesized B. stephanieae ELH peptide into mature individuals induced egg-laying. ELH gene expression in the brain and body was mapped using in-situ hybridization chain reaction. Across the adult brain, 300-400 neurons expressed ELH. Twenty-one different cell types were identified in adults, three of which were located unilaterally on the right side, which corresponds to the location of the reproductive organs. Ten cell types were present in pre-reproductive juvenile stages. An asymmetric cluster of approximately 100 small neurons appeared in the right pedal ganglion of late-stage juveniles. Additional neurons in the pleural and pedal ganglia expressed ELH only in adults that were actively laying eggs and sub-adults that were on the verge of doing so, implicating their direct role in reproduction. Outside the brain, ELH was expressed on sensory appendages, including in presumptive sensory neurons. Its widespread expression in the nudibranch B. stephanieae suggests that ELH plays a role beyond reproduction in gastropod molluscs.

Compiling a Set of Actionable Quality Indicators for Medical Practitioners in Dutch Nursing Homes: A Delphi Study.

Most quality indicators (QIs) currently used in nursing homes reflect the care delivered by the entire multidisciplinary team and are not specific for medical practitioners. International experts have proposed a set of QIs that specifically reflect the quality of medical care in nursing homes. The objective of the Delphi study described here was to compile a set of actionable QIs tailored for medical practitioners working within Dutch nursing homes. This was achieved through the evaluation of 15 existing national QIs and 35 international QIs by a panel of medical practitioners, comprising medical specialists, nurse practitioners, and physician assistants, who are working in Dutch nursing homes. Panelists rated each QI on (1) level of direct control by medical practitioners and (2) its relevance to the quality of medical care. QIs progressing to subsequent rounds required panel agreement on both direct control (≥70% ≥3 points on a 4-point scale) and relevance (≥70% ≥8 on a 10-point scale). In the last round, each panelist selected the 5 most relevant QIs and arranged them in order of importance. These top 5 rankings were converted into points for an overall final ranking. There was consensus on 42 QIs being under the control of medical practitioners, and 21 of these QIs were considered relevant for quality of care. Most of the 21 QIs originated from the international QI set. This finding supports the transferability of the internationally developed QIs to the Dutch nursing home context and provides opportunities to compare the quality of medical care in nursing homes across countries. In the final ranking, the QI related to new medication prescriptions received the highest rating, followed by 3 QIs related to advance care planning. Future research should focus on evaluating the feasibility of measuring the selected QIs and assessing their measurement properties before implementing them in professional learning and quality improvement initiatives for medical practitioners in nursing homes.

Expression patterns and behavioral effects of conopressin and APGWamide in the nudibranch Berghia stephanieae.

The highly conserved oxytocin/vasopressin family of nonapeptides plays many roles across the animal kingdom, from osmoregulation to reproductive physiology. We investigated the expression patterns and pharmacological effects of the gastropod ortholog of this peptide, conopressin, along with another peptide involved in gastropod reproduction, APGWamide, in the nudibranch Berghia stephanieae. A brain transcriptome was used to identify and annotate the gene sequences for the peptides and one conopressin receptor. In-situ hybridization chain reaction showed that many neurons in the brain expressed these peptides. However, the peptide genes were co-expressed by only three neurons, which were in the right cerebral ganglion, the same side on which the reproductive organs are located. A conopressin receptor (BSCPR1) was expressed in a prominent population of APGWamide expressing neurons. Placing animals in a solution containing the APGWamide peptide caused minimal behavioral changes. However, exposure to conopressin reduced locomotion, increased gut contractions, and caused voiding at high concentration. The genes for these peptides and BSCPR1 were expressed in cells in the digestive system. BSCPR1 was also expressed by a line of neurons on the anterior portion of the radula and would be contacted during feeding. APGWamide-expressing neurons were found in the genital ganglion. All three genes expressed in cells on sensory appendages. These results are consistent with the conopressin playing a variety of roles in the brain and the body and being involved in both reproduction and digestion. This study sheds light on the function of this ancient nonapeptide in a new-to-neuroscience invertebrate species.

Recommendations for Next Steps in Our Understanding of Use of Antipsychotics Among Older People in the Post-Acute Long-Term Care Environments: Article 3 of 3.

This article is the third installment of a multi-part series on the history and usage of antipsychotics in older people living in nursing and assisted living facilities. This article presents next steps and recommendations for appropriate usage of antipsychotics in the older population based on the lead author's early drafts, submitted to the editors prior to his untimely death, of this series and on his consultations with the coauthors. Dr Levenson emphasized in his focus on next steps related to antipsychotic use: that all providers should review the history of antipsychotic use and recognize clinically legitimate alternative explanations for the findings. His conclusions were that "off label" usage should not be a reason to exclude the appropriate use of antipsychotics. His overall recommendations to clinicians are to assess and diagnose the underlying cause of the problem, understand the treatment options and select the best one to address the clinical problem and/or the symptom if the problem cannot be fully resolved, and to focus on all medications, not just antipsychotics, in a patient's regimen to aid in a comprehensive understanding of the assessment and inform therapeutic recommendations.

Sample preparation methods for volume electron microscopy in mollusc Berghia stephanieae.

Creating a high-resolution brain atlas in diverse species offers crucial insights into general principles underlying brain function and development. A volume electron microscopy approach to generate such neural maps has been gaining importance due to advances in imaging, data storage capabilities, and data analysis protocols. Sample preparation remains challenging and is a crucial step to accelerate the imaging and data processing pipeline. Here, we introduce several replicable methods for processing the brains of the gastropod mollusc, Berghia stephanieae for volume electron microscopy. Although high-pressure freezing is the most reliable method, the depth of cryopreservation is a severe limitation for large tissue samples. We introduce a BROPA-based method using pyrogallol and methods to rapidly process samples that can save hours at the bench. This is the first report on sample preparation and imaging pipeline for volume electron microscopy in a gastropod mollusc, opening up the potential for connectomic analysis and comparisons with other phyla.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Focusing on Provider Quality Measurement: Continued Consensus and Feasibility Testing of Practice-Based Quality Measures for Primary Care Providers in Long-Term Care.

Medical providers in long-term care (LTC) use a unique skillset in delivering comprehensive resident care. Publicly reported quality measures (QMs) do not directly emphasize medical provider competency and their role in care. The impact of providers is understudied and to a large extent, unknown. Our objective was to define, test, and validate QMs to pragmatically measure the practice-based quality of medical providers in a pilot study. We included 7 North American LTC homes with data from practicing medical providers for LTC residents. We engaged in a 4-phased approach. In phase 1, experts rated 95 candidate QMs using 5 pragmatic-focused criteria in a RAND-modified Delphi process. Phase 2 involved specifying 37 QMs for collection (4 QMs were dropped during pilot testing). We created an abstraction manual and data collection tool for all QMs. Phase 3 involved a retrospective chart review in 7 LTC homes on 33 QMs with trained data abstractors. Data were sufficient to analyze performance for 26 QMs. Lastly, in phase 4 results and psychometric properties were reviewed with an expert panel. They ranked the tested measures for validity and feasibility for use by a nonphysician auditor to evaluate medical provider performance based on medical record review. In total, we examined data from 343 resident charts from 7 LTC homes and 49 providers. Our process yielded 10 QMs as being specified for measurement, feasible to collect, and had good test performance. This is the only study to systematically identify a subset of QMs for feasible collection from the medical record by various data collectors. This pragmatic approach to measuring practice-based quality and quantifying select medical provider competencies allows for the evaluation of individual and facility-level performance and facilitates quality improvement initiatives. Future work should perform broader testing and validate and refine operationalized QMs.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum has long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. The final assembled and filtered Berghia genome is comparable to other high quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes), and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Evolution of cephalopod nervous systems.

Giant brains have independently evolved twice on this planet, in vertebrates and in cephalopods (Figure 1A). Thus, the brains and nervous systems of cephalopods provide an important counterpoint to vertebrates in the search for generalities of brain organization and function. Their mere existence disproves various hypotheses proposed to explain the evolution of the mind and the human brain, such as cognition and large brains evolved only in long-lived animals with complex social systems and parental care, none of which is true of cephalopods. Therefore, it is worthwhile to review what is known about the evolution of cephalopod nervous systems to consider how it informs our understanding of general principles of brain evolution.

State-dependent, visually guided behaviors in the nudibranch Berghia stephanieae.

Nudibranch mollusks have structurally simple eyes whose behavioral roles have not been established. We tested the effects of visual stimuli on the behavior of the nudibranch Berghia stephanieae under different food and hunger conditions. In an arena that was half-shaded, animals spent most of their time in the dark, where they also decreased their speed and made more changes in heading. These behavioral differences between the light and dark were less evident in uniformly illuminated or darkened arenas, suggesting that they were not caused by the level of illumination. Berghia stephanieae responded to distant visual targets; animals approached a black stripe that was at least 15 deg wide on a white background. They did not approach a stripe that was lighter than the background but approached a stripe that was isoluminant with the background, suggesting the detection of spatial information. Animals traveled in convoluted paths in a featureless arena but straightened their paths when a visual target was present even if they did not approach it, suggesting that visual cues were used for navigation. Individuals were less responsive to visual stimuli when food deprived or in the presence of food odor. Thus, B. stephanieae exhibits visually guided behaviors that are influenced by odors and hunger state.

Rates of Hospital-Based Care among Older Adults in the Community and Residential Care Facilities: A Repeated Cross-Sectional Study.

OBJECTIVE: We examine annual rates of emergency department (ED) visits, hospital admissions, and alternate levels of care (ALC) days (ie, the number of days that an older adult remained in hospital when they could not be safely discharged to an appropriate setting in their community) among older adults. DESIGN: Repeated cross-sectional study. SETTING AND PARTICIPANTS: Linked, individual-level health system administrative data on community-dwelling persons, home care recipients, residents of assisted living facilities, and residents of nursing homes aged 65 years and older in Ontario, Canada, from January 1, 2013, to December 31, 2019. METHODS: We calculated rates of ED visits, hospital admissions, and ALC days per 1000 individuals per older adult population per year. We used a generalized linear model with a gaussian distribution, log link, and year fixed effects to obtain rate ratios. RESULTS: There were 1,655,656 older adults in the community, 237,574 home care recipients, 42,600 older adults in assisted living facilities, and 94,055 older adults in nursing homes in 2013; there were 2,129,690 older adults in the community, 281,028 home care recipients, 56,975 older adults in assisted living facilities, and 95,925 older adults in nursing homes in 2019. Residents of assisted living facilities had the highest rates of ED visits (1260.692019 vs 1174.912013), hospital admissions (482.632019 vs 480.192013), and ALC days (1905.572019 vs 1443.032013) per 1000 individuals. Residents of assisted living facilities also had significantly higher rates of ED visits [rate ratio (RR) 3.30, 95% CI 3.20, 3.41), hospital admissions (RR 6.24, 95% CI 6.01, 6.47), and ALC days (RR 25.68, 95% CI 23.27, 28.35) relative to community-dwelling older adults. CONCLUSIONS AND IMPLICATIONS: The disproportionate use of ED visits, hospital admissions, and ALC days among residents of assisted living facilities may be attributed to the characteristics of the population and fragmented licensing and regulation of the sector, including variable models of care. The implementation of interdisciplinary, after-hours, team-based approaches to home and primary care in assisted living facilities may reduce the potentially avoidable use of ED visits, hospital admissions, and ALC days among this population and optimize resource allocation in health care systems.

mEMbrain: an interactive deep learning MATLAB tool for connectomic segmentation on commodity desktops.

Connectomics is fundamental in propelling our understanding of the nervous system's organization, unearthing cells and wiring diagrams reconstructed from volume electron microscopy (EM) datasets. Such reconstructions, on the one hand, have benefited from ever more precise automatic segmentation methods, which leverage sophisticated deep learning architectures and advanced machine learning algorithms. On the other hand, the field of neuroscience at large, and of image processing in particular, has manifested a need for user-friendly and open source tools which enable the community to carry out advanced analyses. In line with this second vein, here we propose mEMbrain, an interactive MATLAB-based software which wraps algorithms and functions that enable labeling and segmentation of electron microscopy datasets in a user-friendly user interface compatible with Linux and Windows. Through its integration as an API to the volume annotation and segmentation tool VAST, mEMbrain encompasses functions for ground truth generation, image preprocessing, training of deep neural networks, and on-the-fly predictions for proofreading and evaluation. The final goals of our tool are to expedite manual labeling efforts and to harness MATLAB users with an array of semi-automatic approaches for instance segmentation. We tested our tool on a variety of datasets that span different species at various scales, regions of the nervous system and developmental stages. To further expedite research in connectomics, we provide an EM resource of ground truth annotation from four different animals and five datasets, amounting to around 180 h of expert annotations, yielding more than 1.2 GB of annotated EM images. In addition, we provide a set of four pre-trained networks for said datasets. All tools are available from https://lichtman.rc.fas.harvard.edu/mEMbrain/. With our software, our hope is to provide a solution for lab-based neural reconstructions which does not require coding by the user, thus paving the way to affordable connectomics.

Clinical Comorbidities and Transitions Between Care Settings Among Residents of Assisted Living Facilities: A Repeated Cross-Sectional Study.

OBJECTIVE: We investigate the changes in the sociodemographic characteristics, clinical comorbidities, and transitions between care settings among residents of assisted living facilities. DESIGN: Repeated cross-sectional study. SETTING AND PARTICIPANTS: Linked, individual-level health system administrative data on residents of assisted living facilities in Ontario, Canada, from January 1, 2013, to December 31, 2019. METHODS: Counts and proportions were calculated to describe the sociodemographic characteristics and clinical comorbidities. Relative changes and trend tests were calculated to quantify the longitudinal changes in the characteristics of residents of assisted living facilities between 2013 and 2019. A Sankey plot was graphed to display transitions between different care settings (ie, hospital admission, nursing home admission, died, or remained in the assisted living facility) each year from 2013 to 2019. RESULTS: There was a 34% relative increase in the resident population size of assisted living facilities (56,9752019 vs 42,6002013). These older adults had a mean age of 87 years, and women accounted for nearly two-thirds of the population across all years. The 5 clinical comorbidities that had the highest relative increases were renal disease (24.3%), other mental health conditions (16.8%), cardiac arrhythmias (9.6%), diabetes (8.5%), and cancer (6.9%). Nearly 20% of the original cohort from 2013 remained in an assisted living facility at the end of 2019, and approximately 10% of that cohort transitioned to a nursing home in any year from 2013 to 2019. CONCLUSIONS AND IMPLICATIONS: Residents of assisted living facilities are an important older adult population that has progressively increased in clinical complexity within less than a decade. Clinicians and policy makers should advocate for the implementation of on-site medical care that is aligned with the needs of these older adults.

Did the Long-Term Care Physician Workforce Change During the Pandemic? Describing MRP Trends in Ontario, Canada.

OBJECTIVES: To examine the practice patterns and trends of long-term care (LTC) physicians between 2019 and 2021 in Ontario, Canada. DESIGN: Population-level descriptive time trend study. SETTING AND PARTICIPANTS: Most responsible physicians (MRPs) of LTC residents of publicly funded LTC homes in Ontario, Canada, from September 2019 to December 2021. METHODS: We examined the number of MRPs in publicly regulated Ontario LTC homes before and during the COVID-19 pandemic using population-level administrative databases. Characteristics of MRPs and practice patterns were generated at baseline and across distinct time periods of the pandemic in descriptive tables. We created a Sankey diagram to visualize MRP practice changes over time. RESULTS: More than one-quarter of pre-pandemic MRPs were no longer MRPs by the end of 2021, although most continued to practice in non-LTC settings. There was a decrease from 1444 to 1266 MRPs over time. Other characteristics of MRPs remained stable over the pandemic time periods. At baseline, LTC physicians were MRP for an average of 57.3 residents. By the end of 2021, this caseload decreased to 53.3 residents per MRP. MRPs increasingly billed monthly management compensation fees over the fee-for-service model across the pandemic time periods. The number of MRPs working in an LTC home shifted to fewer MRPs per home. CONCLUSIONS AND IMPLICATIONS: MRP demographic characteristics did not change over the course of the pandemic. The observed shifts in practice patterns showed a reduction in the overall LTC MRP workforce, who delivered care to fewer residents on average in LTC homes with fewer colleagues to rely on. Future work can study how changes to LTC MRPs' practice patterns impact physician coverage, access and continuity of care, and health services and quality outcomes among residents.

mEMbrain: an interactive deep learning MATLAB tool for connectomic segmentation on commodity desktops.

Connectomics is fundamental in propelling our understanding of the nervous system’s organization, unearthing cells and wiring diagrams reconstructed from volume electron microscopy (EM) datasets. Such reconstructions, on the one hand, have benefited from ever more precise automatic segmentation methods, which leverage sophisticated deep learning architectures and advanced machine learning algorithms. On the other hand, the field of neuroscience at large, and of image processing in particular, has manifested a need for user-friendly and open source tools which enable the community to carry out advanced analyses. In line with this second vein, here we propose mEMbrain, an interactive MATLAB-based software which wraps algorithms and functions that enable labeling and segmentation of electron microscopy datasets in a user-friendly user interface compatible with Linux and Windows. Through its integration as an API to the volume annotation and segmentation tool VAST, mEMbrain encompasses functions for ground truth generation, image preprocessing, training of deep neural networks, and on-the-fly predictions for proofreading and evaluation. The final goals of our tool are to expedite manual labeling efforts and to harness MATLAB users with an array of semi-automatic approaches for instance segmentation. We tested our tool on a variety of datasets that span different species at various scales, regions of the nervous system and developmental stages. To further expedite research in connectomics, we provide an EM resource of ground truth annotation from 4 different animals and 5 datasets, amounting to around 180 hours of expert annotations, yielding more than 1.2 GB of annotated EM images. In addition, we provide a set of 4 pre-trained networks for said datasets. All tools are available from https://lichtman.rc.fas.harvard.edu/mEMbrain/ . With our software, our hope is to provide a solution for lab-based neural reconstructions which does not require coding by the user, thus paving the way to affordable connectomics.

Physician Practice Characteristics Influencing Nurse Practitioner and Physician Assistant Care in Nursing Homes: A Scoping Review.

OBJECTIVES: Physicians, nurse practitioners (NPs), and physician assistants (PAs) are key to the effective delivery of medical care in nursing homes (NHs). Although several studies have reported on the relationship between the care delivered by a given discipline and specific clinical outcomes, the mediating effect of physician practice characteristics is unknown. A scoping review was conducted to determine whether studies examining the impact of NP and PA care in NHs have accounted for both the collaborative relationship between themselves and physicians as well as physician practice size and type. DESIGN: Scoping review. SETTINGS AND PARTICIPANTS: NH and NH residents. METHODS: Papers eligible for review included peer-reviewed studies written in English and that were quantitative in nature. Inclusion criteria required that more than 1 NH and more than a single NP or PA be included for the analysis. Further, the size of the NH studied must have equaled or exceeded 100 residents and more than 1 clinical outcome reported. Databases searched included Ovid MEDLINE, Cochrane Library, Web of Science; CINAHL, and AgeLine. RESULTS: A total of 1878 studies underwent abstract review of which 1719 were excluded. A full-text review of the remaining articles was completed (n = 157, as 2 articles could not be retrieved), of which 16 met eligibility criteria. The study designs were generally retrospective and quasi-experimental in nature. No randomized controlled studies were identified. Physician practice variables such as number of physicians, total practice case load, case mix, and the nature of the collaborative practice between NP/PA and MD were infrequently specified. In no reports was the type of physician practice characterized and no physician practice variables were adjusted for with regard to outcomes. CONCLUSIONS: None of the studies included in the review were found to have incorporated any physician variables into their outcome analyses. Before purporting equivalency between medical provider disciplines in NHs, future studies must, at the very least, consider the mediating effect of physician practice characteristics.