WOMBAT-P: Benchmarking Label-Free Proteomics Data Analysis Workflows.

The inherent diversity of approaches in proteomics research has led to a wide range of software solutions for data analysis. These software solutions encompass multiple tools, each employing different algorithms for various tasks such as peptide-spectrum matching, protein inference, quantification, statistical analysis, and visualization. To enable an unbiased comparison of commonly used bottom-up label-free proteomics workflows, we introduce WOMBAT-P, a versatile platform designed for automated benchmarking and comparison. WOMBAT-P simplifies the processing of public data by utilizing the sample and data relationship format for proteomics (SDRF-Proteomics) as input. This feature streamlines the analysis of annotated local or public ProteomeXchange data sets, promoting efficient comparisons among diverse outputs. Through an evaluation using experimental ground truth data and a realistic biological data set, we uncover significant disparities and a limited overlap in the quantified proteins. WOMBAT-P not only enables rapid execution and seamless comparison of workflows but also provides valuable insights into the capabilities of different software solutions. These benchmarking metrics are a valuable resource for researchers in selecting the most suitable workflow for their specific data sets. The modular architecture of WOMBAT-P promotes extensibility and customization. The software is available at https://github.com/wombat-p/WOMBAT-Pipelines.

The measure of impact: Proposal of quality metrics for solid organ transplant pharmacy practice.

As healthcare continues its transition toward value-based care, it is increasingly important for transplant pharmacists to demonstrate their impact on patient care, health-related outcomes, and healthcare costs. Evidence-based quality and performance metrics are recognized as crucial tools for measuring the value of service. Yet, there is a lack of well-developed and agreed-upon specific metrics for many clinical pharmacy specialties, including solid organ transplantation. To address this need, a panel of transplant pharmacy specialists conducted a detailed literature review and engaged in several panel discussions to identify quality metrics to be considered for assessing the value of clinical pharmacy services provided to solid organ transplant recipients and living donors. The proposed metrics are based on the Donabedian model and are categorized to coincide with the typical phases of transplant care. The measures focus on key issues that arise in transplant recipients related to medication therapy, including adverse drug events, nonadherence, and clinical outcomes attributable to medication therapy management. This article proposes a comprehensive set of measures, any number of which transplant pharmacists can adopt and measure over time to objectively gauge the value of services they are providing to transplant recipients, the transplant center, and the overall healthcare system.

Identifying critical quality metrics in Mohs Surgery: A national expert consensus process.

BACKGROUND: Amid a movement toward value-based healthcare, increasing emphasis has been placed on outcomes and cost of medical services. To define and demonstrate the quality of services provided by Mohs surgeons, it is important to identify and understand the key aspects of Mohs micrographic surgery (MMS) that contribute to excellence in patient care. OBJECTIVE: The purpose of this study is to develop and identify a comprehensive list of metrics in an initial effort to define excellence in MMS. METHODS: Mohs surgeons participated in a modified Delphi process to reach a consensus on a list of metrics. Patients were administered surveys to gather patient perspectives. RESULTS: Twenty-four of the original 66 metrics met final inclusion criteria. Broad support for the initiative was obtained through physician feedback. LIMITATIONS: Limitations of this study include attrition bias across survey rounds and participation at the consensus meeting. Furthermore, the list of metrics is based on expert consensus instead of quality evidence-based outcomes. CONCLUSION: With the goal of identifying metrics that demonstrate excellence in performance of MMS, this initial effort has shown that Mohs surgeons and patients have unique perspectives and can be engaged in a data-driven approach to help define excellence in the field of MMS.

The risk analysis index demonstrates exceptional discrimination in predicting frailty's impact on neurosurgical length of stay quality metrics.

BACKGROUND: Quality measures determine reimbursement rates and penalties in value-based payment models. Frailty impacts these quality metrics across surgical specialties. We compared the discriminatory thresholds for the risk analysis index (RAI), modified frailty index-5 (mFI-5) and increasing patient age for the outcomes of extended length of stay (LOS [eLOS]), prolonged LOS within 30 days (pLOS), and protracted LOS (LOS > 30). METHODS: Patients ≥18 years old who underwent neurosurgical procedures between 2012 and 2020 were queried from the ACS-NSQIP. We performed receiver operating characteristic analysis, and multivariable analyses to examine discriminatory thresholds and identify independent associations. RESULTS: There were 411,605 patients included, with a median age of 59 years (IQR, 48-69), 52.2% male patients, and a white majority 75.2%. For eLOS: RAI C-statistic 0.653 (95% CI: 0.652-0.655), versus mFI-5 C-statistic 0.552 (95% CI: 0.550-0.554) and increasing patient age C-statistic 0.573 (95% CI: 0.571-0.575). Similar trends were observed for pLOS- RAI: 0.718, mFI-5: 0.568, increasing patient age: 0.559, and for LOS>30- RAI: 0.714, mFI-5: 0.548, and increasing patient age: 0.506. Patients with major complications had eLOS 10.1%, pLOS 26.5%, and LOS >30 45.5%. RAI showed a larger effect for all three outcomes, and major complications in multivariable analyses. CONCLUSION: Increasing frailty was associated with three key quality metrics that is, eLOS, pLOS, LOS > 30 after neurosurgical procedures. The RAI demonstrated a higher discriminating threshold compared to both mFI-5 and increasing patient age. Preoperative frailty screening may improve quality metrics through risk mitigation strategies and better preoperative communication with patients and their families.

The global diet quality score as an indicator of adequate nutrient intake and dietary quality - a nation-wide representative study.

BACKGROUND: The Global Diet Quality Score (GDQS) was developed to be a simple, timely and cost-effective tool to track, simultaneously, nutritional deficiency and non-communicable disease risks from diet in diverse settings. The objective was to investigate the performance of GDQS as an indicator of adequate nutrient intake and dietary quality in a national-representative sample of the Brazilian population. METHODS: Nationally-representative data from 44,744 men and non-pregnant and non-lactating women aging ≥ 10 years, from the Brazilian National Dietary Survey were used. Dietary data were collected through two 24-h recalls (24HR). The GDQS was calculated and compared to a proxy indicator of nutrient adequate intake (the Minimum Dietary Diversity for Women-MDD-W) and to an indicator of high-risk diet for non-communicable diseases (caloric contribution from ultra-processed foods-UPF). To estimate the odds for overall nutrient inadequacy across MDD-W and GDQS quintiles, a multiple logistic regression was applied, and the two metrics' performances were compared using Wald's post-test. RESULTS: The mean GDQS for Brazilians was 14.5 (0-49 possible range), and only 1% of the population had a low-risk diet (GDQS ≥ 23). The GDQS mean was higher in women, elderly individuals and in higher-income households. An inverse correlation was found between the GDQS and UPF (rho (95% CI) = -0.20(-0.21;-0.19)). The odds for nutrient inadequacy were lower as quintiles of GDQS and MDD-W were higher (p-trend < 0.001), and MDD-W had a slightly better performance than GDQS (p-diff < 0.001). Having a low-risk GDQS (≥ 23) lowered the odds for nutrient inadequacy by 74% (95% CI:63%-81%). CONCLUSION: The GDQS is a good indicator of overall nutrient adequacy, and correlates well with UPF in a nationally representative sample of Brazil. Future studies must investigate the relationship between the GDQS and clinical endpoints, strengthening the recommendation to use this metric to surveillance dietary risks.

Social workers roles in achieving health quality metrics in primary care: a quality improvement case study.

Value-based payment models may improve patient health by targeting quality of care over quantity of health services. Social workers in primary care settings are well-positioned to improve the quality of health services for vulnerable patients by identifying and addressing patients' social determinants of health. This case study describes a Plan Do Study Act (PDSA) quality improvement approach implemented and refined by social workers to proactively address clinical quality gaps in one family medicine practice. The studied program - entitled Gap Closure Day - was led by a team of social workers to improve quality outcomes of patients. Findings highlight the important roles of social workers as members of health care teams to improve the quality of health services and address health equity.

Author Response.

How to cite this article: Panda BK, Suryawanshi VR, Attarde G, Borkar N, Iyer S, Shah J. Author Response. Indian J Crit Care Med 2024;28(4):408-409.

Performance of the Global Diet Quality Score (GDQS) App in Predicting Nutrient Adequacy and Metabolic Risk Factors among Thai Adults.

BACKGROUND: The Global Diet Quality Score (GDQS) was developed for monitoring nutrient adequacy and diet-related noncommunicable disease risk in diverse populations. A software application (GDQS app) was recently developed for the standardized collection of GDQS data. The application involves a simplified 24-h dietary recall (24HR) where foods are matched to GDQS-food groups using an onboard database, portion sizes are estimated at the food group level using cubic models, and the GDQS is computed. OBJECTIVES: The study aimed to estimate associations between GDQS scores collected using the GDQS app and nutrient adequacy and metabolic risks. METHODS: In this cross-sectional study of 600 Thai males and nonpregnant/nonlactating females (40-60 y), we collected 2 d of GDQS app and paper-based 24HR, food-frequency questionnaires (FFQs), anthropometry, body composition, blood pressure, and biomarkers. Associations between application scores and outcomes were estimated using multiple regression, and application performance was compared with that of metrics scored using 24HR and FFQ data: GDQS, Minimum Dietary Diversity-Women, Alternative Healthy Eating Index-2010, and Global Dietary Recommendations score. RESULTS: In covariate-adjusted models, application scores were significantly (P < 0.05) associated with higher energy-adjusted mean micronutrient adequacy computed using 24HR (range in estimated mean adequacy between score quintiles 1 and 5: 36.3%-44.5%) and FFQ (Q1-Q5: 40.6%-44.2%), and probability of protein adequacy from 24HR (Q1-Q5: 63%-72.5%). Application scores were inversely associated with BMI kg/m2 (Q1-Q5: 26.3-24.9), body fat percentage (Q1-Q5: 31.7%-29.1%), diastolic blood pressure (Q1-Q5: 84-81 mm Hg), and a locally-developed sodium intake score (Q1-Q5: 27.5-24.0 points out of 100); positively associated with high-density lipoprotein cholesterol (Q1-Q5: 49-53 mg/dL) and 24-h urinary potassium (Q1-Q5: 1385-1646 mg); and inversely associated with high midupper arm circumference (Q5/Q1 odds ratio: 0.52) and abdominal obesity (Q5/Q1 odds ratio: 0.51). Significant associations for the application outnumbered those for metrics computed using 24HR or FFQ. CONCLUSIONS: The GDQS app effectively assesses nutrient adequacy and metabolic risk in population surveys.

A prospective study evaluating perioperative quality metrics of cancer surgery: Experience from a tertiary care cancer centre in India.

BACKGROUND: Well-defined quality metrics were studied at this high-volume tertiary care academic oncology centre to evaluate surgical quality and outcomes. METHODS: A prospective cohort of 691 patients undergoing major surgeries for all cancers was observed between 1 September 2018 and 30 August 2019 and predefined quality indicators in three categories: namely, optimal hospital resource utilisation, quality of surgery and perioperative outcomes were recorded during their admission period. Surgical procedures were divided into group I versus group II involving simple/moderate complexity versus high complexity procedures for analysis done using appropriate tests in STATA v14.0. FINDINGS: There were 373 versus 318 patients in group I versus group II with a mean of 48 years of age and 459 (66.43%) being females. Mean BMI was 24.58 kg/m2 , haemoglobin 12.12 g/dL and mean serum albumin of 4.25 g (SD ± 0.6). A total of 667(96.53%) patients had an Eastern Co-Operative Oncology Group performance score of 1 with 271 (39.22%) having comorbidities and 195 (28.22%) having tobacco/alcohol addictions. A total of 393 (56.87%) patients underwent prehabilitation. 83% of patients belonged to lower-middle socioeconomic status and 32% of patients had some prior surgical intervention. Neoadjuvant therapy was administered in 44.5% versus 37.42%, mean preoperative hospital stay was 3.65 versus 6.16 days whereas postoperative was 2.6 versus 6.09 days with total stay being 6.27 versus 12.21 days, mean surgical duration was 132 versus 268 min with mean blood loss of 100 versus 245 mL (group I vs. group II, respectively). Intraoperative events, namely, unexpected bleeding and hypotension occurred in 144 (20.84%) patients while packed red blood cells and/or fresh frozen plasma transfusion was required in 56 (8.1%) patients. 40% of patients needed intensive care unit care postoperatively with 45 (6.51%) patients having some event (hypotension, sepsis, acute kidney injury, etc.). A total of 29 (<4%) patients had ≥grade 3 surgical complications while ≥grade 2 medical complications occurred in 6.43% versus 12.58% of patients (group I vs. group II). A total of 90 (13.02%) patients were readmitted, 15 (2.17%) re-explored and 5 (0.72%) patients died. CONCLUSION: Optimal surgical outcomes in this study compared to international standards underpin the need for well-defined multidisciplinary perioperative pathways to be followed for each organ system and this framework can be adopted by other cancer centres in LMIC to achieve good surgical outcomes.

In health research publications, the number of authors is strongly associated with collective self-citations but less so with citations by others.

OBJECTIVE: . This study investigated the associations between the number of authors and collective self-citations versus citations by others. STUDY DESIGN AND SETTING: . We analyzed 88,594 health science articles published in 2015 and citations they received until 2020. The main variables were the number of authors, the number of citations by co-authors (collective self-citations), and the number of citations by others. RESULTS: . The number of authors correlated more strongly with the number of citations by co-authors than with citations by others (Spearman r 0.31 vs. 0.23; mutually adjusted r 0.26 vs. 0.12). The percentage of self-citations among all citations was 10.6% for single-authored articles, and increased gradually with the number of authors to 34.8% for ≥ 50 authors. Collective self-citations increased the proportion of articles reaching or exceeding 30 total citations by 0.7% for single-authored articles, but by 11.6% for articles written by ≥ 50 authors. CONCLUSIONS: . If citations by others reflect scientific utility, then another mechanism must explain the excess of collective self-citations observed for multi-authored articles. The results support the hypothesis that the authors' own motivations explain this excess. The evaluation of scientific utility should also be based on citations by others, excluding collective self-citations.

Implementing patient safety and quality improvement in dermatology. Part 2: Quality improvement science.

Quality improvement (QI) in medicine is reliant on a team-based approach and an understanding of core QI principles. Part 2 of this continuing medical education series outlines the steps of performing a QI project, from identifying QI opportunities, to carrying out successive Plan-Do-Study-Act cycles, to hard-wiring improvements into the system. QI frameworks will be explored and readers will understand how to interpret basic QI data.

30-day readmission rates among upper gastrointestinal bleeds: A systematic review and meta-analysis.

BACKGROUND AND AIM: Upper gastrointestinal bleeding (UGIB) is a common emergency, with high rates of hospitalization and in-patient mortality compared to other gastrointestinal diseases. Despite readmission rates being a common quality metric, little data are available for UGIBs. This study aimed to determine readmission rates for patients discharged following an UGIB. METHODS: Adhering to PRISMA guidelines, MEDLINE, Embase, CENTRAL, and Web of Science were searched to October 16, 2021. Randomized and non-randomized studies that reported hospital readmission for patients following an UGIB were included. Abstract screening, data extraction, and quality assessment were conducted in duplicate. A random-effects meta-analysis was performed, with statistical heterogeneity measured using I2 . The GRADE framework, with a modified Downs and Black tool, was used to determine certainty of evidence. RESULTS: Seventy studies were included of 1847 screened abstracted, with moderate interrater reliability. Within these studies, 4 292 714 patients were analyzed with a mean age of 66.6 years, and 54.7% male. UGIB had a 30-day all-cause readmission rate of 17.4% (95% confidence interval [CI] 16.7-18.2%), stratification revealed a higher rate for variceal UGIB [19.6% (95% CI 17.6-21.5%)] than non-variceal [16.8% (95% CI 16.0-17.5%)]. Only one third were readmitted due to recurrent UGIB (4.8% [95% CI 3.1-6.4%]). UGIB due to peptic ulcer bleeding had the lowest 30-day readmission rate [6.9% (95% CI 3.8-10.0%)]. Certainty of evidence was low or very low for all outcomes. CONCLUSIONS: Almost one in five patients discharged after an UGIB are readmitted within 30 days. These data should prompt clinicians to reflect on their own practice to identify areas of strength or improvement.

Nova diet quality scores and risk of weight gain in the NutriNet-Brasil cohort study.

OBJECTIVE: To assess the prospective association of two diet quality scores based on the Nova food classification with BMI gain. DESIGN: The NutriNet-Brasil cohort is an ongoing web-based prospective study with continuous recruitment of participants aged ≥ 18 years since January 2020. A short 24-h dietary recall screener including 'yes/no' questions about the consumption of whole plant foods (WPF) and ultra-processed foods (UPF) was completed by participants at baseline. The Nova-WPF and the Nova-UPF scores were computed by adding up positive responses regarding the consumption of thirty-three varieties of WPF and twenty-three varieties of UPF, respectively. Participants reported their height at baseline and their weight at both baseline and after approximately 15 months of follow-up. A 15-month BMI (kg/m2) increase of ≥5 % was coded as BMI gain. SETTING: Brazil. PARTICIPANTS: 9551 participants from the NutriNet-Brasil cohort. RESULTS: Increasing quintiles of the Nova-UPF score were linearly associated with higher risk of BMI gain (relative risk Q5/Q1 = 1·34; 95 % CI 1·15, 1·56), whereas increasing quintiles of the Nova-WPF score were linearly associated with lower risk (relative risk Q5/Q1 = 0·80; 95 % CI 0·69, 0·94). We identified a moderate inverse correlation between the two scores (-0·33) and a partial mediating effect of the alternative score: 15 % for the total effect of the Nova-UPF score and 25 % for the total effect of the Nova-WPF score. CONCLUSIONS: The Nova-UPF and Nova-WPF scores are independently associated with mid-term BMI gain further justifying their use in diet quality monitoring systems.

Impact of Telemedicine Modality on Quality Metrics in Diverse Settings: Implementation Science-Informed Retrospective Cohort Study.

BACKGROUND: Video-based telemedicine (vs audio only) is less frequently used in diverse, low socioeconomic status settings. Few prior studies have evaluated the impact of telemedicine modality (ie, video vs audio-only visits) on clinical quality metrics. OBJECTIVE: The aim of this study was to assess telemedicine uptake and impact of visit modality (in-person vs video and phone visits) on primary care quality metrics in diverse, low socioeconomic status settings through an implementation science lens. METHODS: Informed by the RE-AIM (Reach, Effectiveness, Adoption, Implementation, and Maintenance) framework, we evaluated telemedicine uptake, assessed targeted primary care quality metrics by visit modality, and described provider-level qualitative feedback on barriers and facilitators to telemedicine implementation. RESULTS: We found marginally better quality metrics (ie, blood pressure and depression screening) for in-person care versus video and phone visits; de-adoption of telemedicine was marked within 2 years in our population. CONCLUSIONS: Following the widespread implementation of telemedicine during the COVID-19 pandemic, the impact of visit modality on quality outcomes, provider and patient preferences, as well as technological barriers in historically marginalized settings should be considered.

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories.

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes.

Gaussian of Differences: A Simple and Efficient General Image Fusion Method.

The separate analysis of images obtained from a single source using different camera settings or spectral bands, whether from one or more than one sensor, is quite difficult. To solve this problem, a single image containing all of the distinctive pieces of information in each source image is generally created by combining the images, a process called image fusion. In this paper, a simple and efficient, pixel-based image fusion method is proposed that relies on weighting the edge information associated with each pixel of all of the source images proportional to the distance from their neighbors by employing a Gaussian filter. The proposed method, Gaussian of differences (GD), was evaluated using multi-modal medical images, multi-sensor visible and infrared images, multi-focus images, and multi-exposure images, and was compared to existing state-of-the-art fusion methods by utilizing objective fusion quality metrics. The parameters of the GD method are further enhanced by employing the pattern search (PS) algorithm, resulting in an adaptive optimization strategy. Extensive experiments illustrated that the proposed GD fusion method ranked better on average than others in terms of objective quality metrics and CPU time consumption.

Quality Metrics in Acute Stroke: Time to Own.

How to cite this article: Ahmed A. Quality Metrics in Acute Stroke: Time to Own. Indian J Crit Care Med 2023;27(11):786-787.

Correlation of Quality Metrics of Acute Stroke Care with Clinical Outcomes in an Indian Tertiary-care University Hospital: A Prospective Evidence-based Study.

Aim: To characterize the impact of adherence to quality metrics of stroke care on the clinical outcomes of ischemic stroke (IS) and intracerebral hemorrhage (ICH) admissions. Methods: Consecutive patients with acute stroke were prospectively followed up for their demographic and clinical characteristics, acute stroke management, and associated clinical outcomes at discharge. Stroke quality metrics [adopted from the American Heart Association (AHA)/American Stroke Association's Get with The Guidelines (GWTG)] with a specific interest in an association between acute reperfusion therapies and functional recovery in stroke patients are analyzed and presented. A composite measure of care was considered "0 (non-adherence) to 1 (adherence)." An all-or-none measure of care was calculated to check whether eligible patients received all the quality-of-care interventions. Multivariate Cox regression models were used to study an association between optimal adherence and clinical outcomes. Results: During the study period, of the total 256 stroke admissions, 200 (78.1%) patients had IS, and the remaining 56 (21.9%) patients had ICH. The median [interquartile range (IQR)] age of total stroke admissions was 57 (36-78) years. Male preponderance was observed (IS: 80% and ICH: 67.9%). The conformity of performance metrics in IS patients was from 69.1% [95% confidence interval (CI), 68.5-69.6] for the use of deep vein thrombosis prophylaxis (DVTp) to 97.8% (95% CI, 96.2-98.6) for the use of statins. In ICH patients, it ranged from 61.7% (95% CI, 60.4-62.5) for the use of DVTp to 89.9% (95% CI, 88.6-89.7) for stroke rehabilitation. The unadjusted odds ratio (OR) of mortality (in-hospital plus the 28th-day postdischarge) was higher in ICH patients vs IS patients (4.42, p = 0.005). Optimal adherence with intravenous recombinant tissue plasminogen activator (IV-rtPA) therapy [hazards ratio (HR) = 0.23], in-hospital acute measures [IS (HR = 0.41) and ICH (HR = 0.63)], and discharge measures [IS (HR = 0.35) and ICH (HR = 0.45)] were associated with reduced hazards of the 28th-day mortality in both cohorts. Compared to ICH, IS patients had significantly improved neurofunctional recovery [modified Rankin score (mRS) ≤ 2, p < 0.01]. Conclusion: Adherence to quality metrics and performance measures was associated with low mortality and favorable clinical outcomes. Also, DVTp as an in-hospital (acute) measure of stroke care needs attention in both cerebrovascular events. How to cite this article: Panda BK, Suryawanshi VR, Attarde G, Borkar N, Iyer S, Shah J. Correlation of Quality Metrics of Acute Stroke Care with Clinical Outcomes in an Indian Tertiary-care University Hospital: A Prospective Evidence-based Study. Indian J Crit Care Med 2023;27(11):806-815.

MEPSi: A tool for simulating tomograms of membrane-embedded proteins.

The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (Membrane Embedded Protein Simulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane αIIbß3 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins.

Using single-worm RNA sequencing to study C. elegans responses to pathogen infection.

BACKGROUND: Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. RESULTS: In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. CONCLUSIONS: We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.