Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.

Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.

Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH.

Imaging the transcriptome in situ with high accuracy has been a major challenge in single-cell biology, which is particularly hindered by the limits of optical resolution and the density of transcripts in single cells1-5. Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells-with high accuracy and sub-diffraction-limit resolution-in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope. The transcriptome-level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand-receptor pairs across neighbouring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ.

Excess mortality from COVID and non-COVID causes in minority populations.

The 2020 US mortality totaled 2.8 million after early March, which is 17.3% higher than age-population-weighted mortality over the same time interval in 2017 to 2019, for a total excess death count of 413,592. We use data on weekly death counts by cause, as well as life tables, to quantify excess mortality and life years lost from both COVID-19 and non-COVID-19 causes by race/ethnicity, age, and gender/sex. Excess mortality from non-COVID-19 causes is substantial and much more heavily concentrated among males and minorities, especially Black, non-Hispanic males, than COVID-19 deaths. Thirty-four percent of the excess life years lost for males is from non-COVID-19 causes. While minorities represent 36% of COVID-19 deaths, they represent 70% of non-COVID-19 related excess deaths and 58% of non-COVID-19 excess life years lost. Black, non-Hispanic males represent only 6.9% of the population, but they are responsible for 8.9% of COVID-19 deaths and 28% of 2020 excess deaths from non-COVID-19 causes. For this group, nearly half of the excess life years lost in 2020 are due to non-COVID-19 causes.

The effects of health facility access and quality on family planning decisions in urban Senegal.

Research in developing countries is rarely focused on examining how supply side factors affect family planning decisions due to a lack of facility-level data. When these data exist, analyses tend to focus on rural environments. In this paper, we study the effects that health facility access and quality have on contraceptive use and desired number of children for women in urban Senegal. Unlike related studies focusing on rural environments, we find no evidence that greater access to health facilities and pharmacies increases contraceptive use among urban women. However, we do find that contraceptive use among urban women is higher with greater facility quality. For example, we find that increasing the proportion of pharmacies employing multiple pharmacists from 0% to 50% would increase contraceptive use by 6.0 percentage points, and increasing the proportion of facilities with family planning guidelines/protocols from 50% to 100% would increase use by 2.1 percentage points.

Systematic profiling of Caenorhabditis elegans locomotive behaviors reveals additional components in G-protein Gαq signaling.

Genetic screens have been widely applied to uncover genetic mechanisms of movement disorders. However, most screens rely on human observations of qualitative differences. Here we demonstrate the application of an automatic imaging system to conduct a quantitative screen for genes regulating the locomotive behavior in Caenorhabditis elegans. Two hundred twenty-seven neuronal signaling genes with viable homozygous mutants were selected for this study. We tracked and recorded each animal for 4 min and analyzed over 4,400 animals of 239 genotypes to obtain a quantitative, 10-parameter behavioral profile for each genotype. We discovered 87 genes whose inactivation causes movement defects, including 50 genes that had never been associated with locomotive defects. Computational analysis of the high-content behavioral profiles predicted 370 genetic interactions among these genes. Network partition revealed several functional modules regulating locomotive behaviors, including sensory genes that detect environmental conditions, genes that function in multiple types of excitable cells, and genes in the signaling pathway of the G protein Gαq, a protein that is essential for animal life and behavior. We developed quantitative epistasis analysis methods to analyze the locomotive profiles and validated the prediction of the γ isoform of phospholipase C as a component in the Gαq pathway. These results provided a system-level understanding of how neuronal signaling genes coordinate locomotive behaviors. This study also demonstrated the power of quantitative approaches in genetic studies.

Eosinophilic Enteritis Causing Recurrent Small Bowel Obstruction: A Case Report.

Eosinophilic enteritis is an inflammatory condition characterized by eosinophilic infiltration of the gastrointestinal tract. This case report highlights a unique presentation of eosinophilic enteritis as a cause of recurrent small bowel obstruction. The diagnosis was elusive despite extensive abdominal imaging. A histopathologic examination of a full-thickness bowel segment showing extensive eosinophilic infiltration in the muscularis propria was vital in establishing the diagnosis. This report underscores the diagnostic complexities associated with eosinophilic enteritis and the need to consider this condition as a potential cause of recurrent abdominal pain and small bowel obstruction.

The demand for skills training among Medicaid home-based caregivers.

Medicaid spends nearly 100 billion dollars annually on home and community-based care for the disabled. Much of this care is provided by personal care aides, few of whom have received training related to the services they provide. We conducted a randomized controlled trial to estimate their demand for training. We find that 13 percent of these caregivers complete training without an incentive. Paying the caregivers four times their hourly wage increases training completion by roughly nine percentage points. Additional experimental variation suggests that among individuals confirmed to be aware of the training, the financial incentive increases completion from 35 to 58 percent. Demand curves based on these results suggest that while many caregivers value the opportunity to train, policies aimed at universal take up require large financial incentives.

Real-world outcomes and toxicity of adjuvant chemotherapy in NSCLC: a single-center experience.

Aim: Adjuvant chemotherapy in NSCLC is associated with modest benefits and significant toxicity. We sought to evaluate the toxicity of adjuvant chemotherapy and disease-specific outcomes in a real-world population. Methods: We performed a retrospective analysis of patients undergoing adjuvant chemotherapy for NSCLC in an Irish center over a 7-year period. We described treatment-associated toxicity, recurrence-free survival and overall survival. Results: 62 patients underwent adjuvant chemotherapy. Treatment-associated hospitalisation occurred in 29% of patients. Relapse was recorded in 56% of patients and median recurrence-free survival was 27 months. Conclusion: High rates of disease recurrence and treatment-associated morbidity were observed in patients receiving adjuvant chemotherapy for NSCLC. Novel therapeutic strategies are required to improve outcomes in this population.

Early NSCLC is primarily managed with lung cancer surgery. Chemotherapy is offered to some patients with early NSCLC after surgery depending on the size of the tumor and lymph node involvement. The benefit of post-surgery (adjuvant) chemotherapy in reducing the chance of cancer recurrence has been shown to be relatively small in previous studies. Large studies of post-surgery chemotherapy in NSCLC have included limited numbers of older patients, and patients with significant medical issues. This study looked to evaluate the safety and treatment-associated side effects of adjuvant chemotherapy in a group of patients more reflective of everyday clinical practice, and to assess the rates of cancer recurrence in these patients. In our study of 62 patients who underwent adjuvant chemotherapy for NSCLC, nearly 1 in 3 patients required hospital admission due to treatment-associated side effects. Almost 40% of patients experienced significant blood test abnormalities (including anaemia, reduced platelets, and reduced white blood cells). Approximately 1 in 4 patients required a reduction in the doses of their treatment due to treatment-associated side effects. More than half of the patients in our study experienced a recurrence of their cancer. Our study demonstrates the significant side effects associated with adjuvant chemotherapy in NSCLC and highlights the need for better-tolerated treatment strategies to reduce cancer recurrence in early NSCLC.

Nursing home quality, COVID-19 deaths, and excess mortality.

The COVID-19 pandemic in the US has been particularly devastating for nursing home residents. A key question is how have some nursing homes been able to effectively protect their residents, while others have not? Using data on the universe of US nursing homes, we examine whether establishment quality is predictive of COVID-19 mortality. Higher-quality nursing homes, as measured by CMS overall five-star rating, have substantially lower COVID-19 mortality through September of 2020. Quality does not predict the ability to prevent any COVID-19 resident or staff cases, but higher-quality establishments prevent the spread of resident infections conditional on having one. Preventing COVID-19 cases and deaths may come at some cost, as high-quality homes have substantially higher non-COVID deaths. The positive correlation between establishment quality and non-COVID mortality is strong enough that high-quality homes also have more total deaths than their low-quality counterparts and this relationship has grown with time. As of late April 2021, five-star homes have experienced 8.4 percent more total deaths than one-star homes.

Case report: BRAF-inhibitor therapy in BRAF-mutated primary CNS tumours including one case of BRAF-mutated Rosai-Dorfman disease.

BRAF V600E oncogene mutations have been reported in multiple central nervous system (CNS) tumor types, and emerging evidence supports the use of targeted therapy in BRAF-mutated gliomas. BRAF oncogene mutations have been recently identified in Rosai-Dorfman disease (RDD)-a rare non-Langerhans cell histiocytosis. This series describes three patients from two neurosurgical centers in Ireland with BRAF V600E-mutated CNS tumors. The study participants include a 19-year-old male patient with ganglioglioma with anaplastic features, a 21-year-old male patient with CNS involvement of RDD, and a 28-year-old female patient with ganglioglioma with anaplastic features. Two patients received radiation with concurrent temozolomide before BRAF-targeted therapy. This case series describes clinical and radiological responses to BRAF-targeted therapy in BRAF V600E-mutated gliomas across multiple tumor grades and is only the second published report of response to targeted therapy in BRAF-mutated RDD. The durability of disease control with BRAF-targeted therapy was generally superior to that achieved with chemoradiation; one patient has experienced ongoing disease control for 5 years. The reported case of treatment response in BRAF-mutated RDD supports the strategy of genotyping and utilization of targeted therapy in this rare disease. The optimal sequencing of BRAF-targeted therapy in BRAF-mutated gliomas/glioneuronal tumors remains unclear, and further prospective studies are required to guide the use of genome-matched therapy in this patient population.

Total shutdowns, targeted restrictions, or individual responsibility: How to promote social distancing in the COVID-19 Era?

We examine the impact of early state and local COVID-19 policies to encourage social distancing. Outcomes are daily foot traffic at establishments spanning ten key industries, across which transmission risk varies substantially. Policies include state of emergency declarations, blunt general restrictions such as stay-at-home (SAH) orders, and targeted rules such as restrictions on bars, restaurants, entertainment venues, and schools. Exploiting variation in the timing of policies in difference-in-difference models, we show that much of the decline in foot traffic early in the pandemic was due to private precautionary behavior. SAH orders explain almost none of the foot traffic decline in industries with high risk of virus transmission, but they do explain a substantial share of the decline in moderate- to low-risk industries such as outdoor sports and visits to parks. Targeted restrictions tend to impact intended industries, as well as complementary ones. We show that the impact of targeted restrictions is largest in counties with no SAH restrictions, suggesting that better targeting of public restrictions can have important efficiency gains.

Employment effects of healthcare policy: Evidence from the 2007 FDA black box warning on antidepressants.

Public policies aimed at improving health may have indirect effects on outcomes such as education and employment. We study the labor market effects of the US Food and Drug Administration's 2007 expanded black box warning on antidepressants. Our difference-in-differences estimates imply that the warning reduced employment by 6.1 percent among women aged 35-49 with a history of depression. We explore potential mechanisms and find that antidepressant and psychotherapy use among women aged 35-49 decreased after the warning. Our analysis suggests that the 2007 warning reduced US labor force participation by 0.23 percentage points, leading to $11.8 billion in lost wages.

Measurement Error in Discrete Health Facility Choice Models: an Example from Urban Senegal.

We use individual-level health facility choice data from urban Senegal to estimate consumer preferences for facility characteristics related to maternal health services. We find that consumers consider a large number of quality related facility characteristics, as well as travel costs, when making their health facility choice. In contrast to the typical assumption in the literature, our findings indicate that individuals frequently bypass the facility nearest their home. In light of this, we show that the mismeasured data used commonly in the literature produces biased preference estimates; most notably, the literature likely overestimates consumer distaste for travel.

Systems level circuit model of C. elegans undulatory locomotion: mathematical modeling and molecular genetics.

To establish the relationship between locomotory behavior and dynamics of neural circuits in the nematode C. elegans we combined molecular and theoretical approaches. In particular, we quantitatively analyzed the motion of C. elegans with defective synaptic GABA and acetylcholine transmission, defective muscle calcium signaling, and defective muscles and cuticle structures, and compared the data with our systems level circuit model. The major experimental findings are: (1) anterior-to-posterior gradients of body bending flex for almost all strains both for forward and backward motion, and for neuronal mutants, also analogous weak gradients of undulatory frequency, (2) existence of some form of neuromuscular (stretch receptor) feedback, (3) invariance of neuromuscular wavelength, (4) biphasic dependence of frequency on synaptic signaling, and (5) decrease of frequency with increase of the muscle time constant. Based on (1) we hypothesize that the Central Pattern Generator (CPG) is located in the head both for forward and backward motion. Points (1) and (2) are the starting assumptions for our theoretical model, whose dynamical patterns are qualitatively insensitive to the details of the CPG design if stretch receptor feedback is sufficiently strong and slow. The model reveals that stretch receptor coupling in the body wall is critical for generation of the neuromuscular wave. Our model agrees with our behavioral data (3), (4), and (5), and with other pertinent published data, e.g., that frequency is an increasing function of muscle gap-junction coupling.

The effects of prospective mate quality on investments in healthy body weight among single women.

This paper examines how a single female's investment in healthy body weight is affected by the quality of single males in her marriage market. A principle concern in estimation is the presence of market-level unobserved heterogeneity that may be correlated with changes in single male quality, measured as earning potential. To address this concern, we employ a differencing strategy that normalizes the exercise behaviors of single women to those of their married counterparts. Our main results suggest that when potential mate quality in a marriage market decreases, single black women invest less in healthy body weight. For example, we find that a 10 percentage point increase in the proportion of low quality single black males leads to a 5-10% decrease in vigorous exercise taken by single black females. Results for single white women are qualitatively similar, but not consistent across specifications. These results highlight the relationship between male and female human capital acquisition that is driven by participation in the marriage market. Our results suggest that programs designed to improve the economic prospects of single males may yield positive externalities in the form of improved health behaviors, such as more exercise, particularly for single black females.

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

Automated imaging of C. elegans behavior.

Automated systems for recording and analyzing behavior have many applications for the study of neurobiology in Caenorhabditis elegans. In particular, machine-based approaches allow for precise quantitative definitions of behavioral phenotypes that have traditionally been subjectively described by individual observers. Automated systems also facilitate the analysis of behaviors that occur over long time scales or are difficult to detect by eye. Here we describe the detailed methodology for the use of one recently described automated tracking system for C. elegans. These protocols make it possible to measure a wide range of parameters related to the morphology, body posture, and locomotion patterns of individual wild-type and mutant nematodes.

An automated system for measuring parameters of nematode sinusoidal movement.

BACKGROUND: Nematode sinusoidal movement has been used as a phenotype in many studies of C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior. RESULTS: We have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worm's centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements. To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly (activation of Gq-alpha versus loss of Go-alpha function), as well as of different mutant alleles at a single locus (null and dominant negative alleles of the goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb. CONCLUSIONS: Automated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.

An imaging system for standardized quantitative analysis of C. elegans behavior.

BACKGROUND: The nematode Caenorhabditis elegans is widely used for the genetic analysis of neuronal cell biology, development, and behavior. Because traditional methods for evaluating behavioral phenotypes are qualitative and imprecise, there is a need for tools that allow quantitation and standardization of C. elegans behavioral assays. RESULTS: Here we describe a tracking and imaging system for the automated analysis of C. elegans morphology and behavior. Using this system, it is possible to record the behavior of individual nematodes over long time periods and quantify 144 specific phenotypic parameters. CONCLUSIONS: These tools for phenotypic analysis will provide reliable, comprehensive scoring of a wide range of behavioral abnormalities, and will make it possible to standardize assays such that behavioral data from different labs can readily be compared. In addition, this system will facilitate high-throughput collection of phenotypic data that can ultimately be used to generate a comprehensive database of C. elegans phenotypic information. AVAILABILITY: The hardware configuration and software for the system are available from wschafer@ucsd.edu.

Exploring variability and sources of measurement error in alcohol expectancy questionnaire reliability coefficients: a meta-analytic reliability generalization study.

OBJECTIVE: Reliability Generalization, a relatively new meta-analytic score reliability technique, was used to examine reliability coefficients for Alcohol Expectancy Questionnaire (AEQ) subscales in published research. Specifically, the present study identified the typical reliability coefficients of scores on AEQ subscales across published studies and examined sources of measurement error across AEQ subscales and studies. METHOD: Based on literature reviews of PsyclNFO and PubMed, a total of 71 studies were analyzed and coded on 10 different dimensions believed to affect score reliability. RESULTS: Only 37% of the studies surveyed reported reliability coefficients for the data in hand. Analysis of these studies revealed that the average score reliability across studies varied considerably in subscales and samples, with the Arousal/Interpersonal Power and Sexual Enhancement subscales evidencing the greatest variability. Gender homogeneity and racial homogeneity were found to be the two most important predictors of the magnitude of score reliability coefficients across subscales and studies. CONCLUSIONS: Results of the study indicate that the AEQ tends to generate reliable scores, with some noted exceptions. Because the Arousal/Interpersonal Power and Sexual Enhancement subscales tend to generate unacceptably low reliability coefficients and because the AEQ holds promise in furthering prevention and treatment outcome research, it is suggested that future research with the AEQ explore instrument factor structure and item consistency. Because reliability estimates can influence substantive statistical analyses, it is recommended that authors using the AEQ report reliability information in their published work.