Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Mechanisms of Cell Death Induced by Erastin in Human Ovarian Tumor Cells.

Erastin (ER) induces cell death through the formation of reactive oxygen species (ROS), resulting in ferroptosis. Ferroptosis is characterized by an accumulation of ROS within the cell, leading to an iron-dependent oxidative damage-mediated cell death. ER-induced ferroptosis may have potential as an alternative for ovarian cancers that have become resistant due to the presence of Ras mutation or multi-drug resistance1 (MDR1) gene expression. We used K-Ras mutant human ovarian tumor OVCAR-8 and NCI/ADR-RES, P-glycoprotein-expressing cells, to study the mechanisms of ER-induced cell death. We used these cell lines as NCI/ADR-RES cells also overexpresses superoxide dismutase, catalase, glutathione peroxidase, and transferase compared to OVCAR-8 cells, leading to the detoxification of reactive oxygen species. We found that ER was similarly cytotoxic to both cells. Ferrostatin, an inhibitor of ferroptosis, reduced ER cytotoxicity. In contrast, RSL3 (RAS-Selective Ligand3), an inducer of ferroptosis, markedly enhanced ER cytotoxicity in both cells. More ROS was detected in OVCAR-8 cells than NCI/ADR-RES cells, causing more malondialdehyde (MDA) formation in OVCAR-8 cells than in NCI/ADR-RES cells. RSL3, which was more cytotoxic to NCI/ADR-RES cells, significantly enhanced MDA formation in both cells, suggesting that glutathione peroxidase 4 (GPX4) was involved in ER-mediated ferroptosis. ER treatment modulated several ferroptosis-related genes (e.g., CHAC1, GSR, and HMOX1/OX1) in both cells. Our study indicates that ER-induced ferroptotic cell death may be mediated similarly in both NCI/ADR-RES and OVCAR-8 cells. Additionally, our results indicate that ER is not a substrate of P-gp and that combinations of ER and RSL3 may hold promise as more effective treatment routes for ovarian cancers, including those that are resistant to other current therapeutic agents.

Substratum stiffness tunes membrane voltage in mammary epithelial cells.

Membrane voltage (Vm) plays a critical role in the regulation of several cellular behaviors, including proliferation, apoptosis and phenotypic plasticity. Many of these behaviors are affected by the stiffness of the underlying extracellular matrix, but the connections between Vm and the mechanical properties of the microenvironment are unclear. Here, we investigated the relationship between matrix stiffness and Vm by culturing mammary epithelial cells on synthetic substrata, the stiffnesses of which mimicked those of the normal mammary gland and breast tumors. Although proliferation is associated with depolarization, we surprisingly observed that cells are hyperpolarized when cultured on stiff substrata, a microenvironmental condition that enhances proliferation. Accordingly, we found that Vm becomes depolarized as stiffness decreases, in a manner dependent on intracellular Ca2+. Furthermore, inhibiting Ca2+-gated Cl- currents attenuates the effects of substratum stiffness on Vm. Specifically, we uncovered a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of Vm by substratum stiffness. Taken together, these results suggest a novel role for CFTR and membrane voltage in the response of mammary epithelial cells to their mechanical microenvironment.

Which Road to Recovery?: Factors Influencing Postacute Stroke Discharge Destinations: A Delphi Study.

BACKGROUND AND PURPOSE: The criteria for determining the level of postacute care for patients with stroke are variable and inconsistent. The purpose of this study was to identify key factors influencing the selection of postacute level of care for these patients. METHODS: We used a collaborative 4-round Delphi process to achieve a refined list of factors influencing postacute level of care selection. Our Delphi panel of experts consisted of 32 panelists including physicians, physical therapists, occupational therapists, speech-language pathologists, nurses, stroke survivors, administrators, policy experts, and individuals associated with third-party insurance companies. RESULTS: In round 1, 207 factors were proposed, with subsequent discussion resulting in consolidation into 15 factors for consideration. In round 2, 15 factors were ranked with consensus on 10 factors; in round 3,10 factors were ranked with consensus on 9 factors. In round 4, the final round, 9 factors were rated with Likert scores ranging from 5 (most important) to 1(not important). The percentage of panelists who provided a rating of 4 or above were as follows: likelihood to benefit from an active rehabilitation program (97%), need for clinicians with specialized rehabilitation skills (94%), need for active and ongoing medical management and monitoring (84%), ability to tolerate an active rehabilitation program (74%), need for caregiver training to return to the community (48%), family/caregiver support (39%), likelihood to return to community/home (39%), ability to return to physical home environment (32%), and premorbid dementia (16%). CONCLUSIONS: This study provides an expert, consensus-based set of key factors to be considered when determining where stroke patients are discharged for postacute care. These factors may be useful in developing a decision support tool for use in clinical settings.

Whole-Genome Sequencing Association Analyses of Stroke and Its Subtypes in Ancestrally Diverse Populations From Trans-Omics for Precision Medicine Project.

BACKGROUND AND PURPOSE: Stroke is the leading cause of death and long-term disability worldwide. Previous genome-wide association studies identified 51 loci associated with stroke (mostly ischemic) and its subtypes among predominantly European populations. Using whole-genome sequencing in ancestrally diverse populations from the Trans-Omics for Precision Medicine (TOPMed) Program, we aimed to identify novel variants, especially low-frequency or ancestry-specific variants, associated with all stroke, ischemic stroke and its subtypes (large artery, cardioembolic, and small vessel), and hemorrhagic stroke and its subtypes (intracerebral and subarachnoid). METHODS: Whole-genome sequencing data were available for 6833 stroke cases and 27 116 controls, including 22 315 European, 7877 Black, 2616 Hispanic/Latino, 850 Asian, 54 Native American, and 237 other ancestry participants. In TOPMed, we performed single variant association analysis examining 40 million common variants and aggregated association analysis focusing on rare variants. We also combined TOPMed European populations with over 28 000 additional European participants from the UK BioBank genome-wide array data through meta-analysis. RESULTS: In the single variant association analysis in TOPMed, we identified one novel locus 13q33 for large artery at whole-genome-wide significance (P<5.00×10-9) and 4 novel loci at genome-wide significance (P<5.00×10-8), all of which need confirmation in independent studies. Lead variants in all 5 loci are low-frequency but are more common in non-European populations. An aggregation of synonymous rare variants within the gene C6orf26 demonstrated suggestive evidence of association for hemorrhagic stroke (P<3.11×10-6). By meta-analyzing European ancestry samples in TOPMed and UK BioBank, we replicated several previously reported stroke loci including PITX2, HDAC9, ZFHX3, and LRCH1. CONCLUSIONS: We represent the first association analysis for stroke and its subtypes using whole-genome sequencing data from ancestrally diverse populations. While our findings suggest the potential benefits of combining whole-genome sequencing data with populations of diverse genetic backgrounds to identify possible low-frequency or ancestry-specific variants, they also highlight the need to increase genome coverage and sample sizes.

Association of Baseline Cardiac Troponin With Acute Myocardial Infarction in Stroke Patients Presenting Within 4.5 Hours.

Background and Purpose- American Heart Association guidelines recommend obtaining baseline troponin in all patients with acute ischemic stroke. Yet, there is a paucity of data on the prevalence of baseline troponin elevation and specifically its diagnostic yield for acute myocardial infarction (AMI) in patients presenting within the time window for thrombolysis. Methods- We retrospectively analyzed 1072 consecutive patients admitted for acute ischemic stroke or transient ischemic attack, who presented within 4.5 hours of last known well (LKW). Patients who had baseline cardiac troponin I (bcTnI) obtained within 72 hours from LKW (n=525) were included in the study. Multivariable logistic regression was conducted to determine factors independently related to an elevated bcTnI (>0.04 ng/mL). We calculated the area under receiver operator curves, sensitivity, and specificity, to determine the diagnostic accuracy of (i) the bcTnI for AMI stratified by the time to assessment and (ii) the best time cutoff for obtaining bcTnI. Results- Among included subjects, the median time from LKW to the bcTnI was 3.8 hours and 113 (21.5%) subjects had an elevated bcTnI. Assessment of bcTnI within 4.5 hours from LKW was significantly more often associated with normal values as compared to assessment between 4.5 and 72 hours (61.7% versus 38.3%; P=0.001). Fifteen (2.9%) patients were diagnosed with AMI. After adjustment for pertinent confounders, time to bcTnI assessment was independently associated with AMI (odds ratio, 1.04 [95% CI, 1.02-1.07] P=0.001). When stratified by time, bcTnI assessed within 4.5 hours had a sensitivity of 25% and specificity of 83.7% for AMI, whereas bcTnI assessment between 4.5 and 72 hours was associated with a sensitivity of 90.9% and specificity of 74.8%. Conclusions- Assessment of bcTnI after 4.5 hours from LKW was associated with greater diagnostic accuracy than testing within 4.5 hours. This information may inform routine clinical practice.

Redefined Measure of Early Neurological Improvement Shows Treatment Benefit of Alteplase Over Placebo.

Background and Purpose- The first of the 2 NINDS (National Institute of Neurological Disorders and Stroke) Study trials did not show a significant increase in early neurological improvement, defined as National Institutes of Health Stroke Scale (NIHSS) improvement by ≥4, with alteplase treatment. We hypothesized that early neurological improvement defined as a percentage change in NIHSS (percent change NIHSS) at 24 hours is superior to other definitions in predicting 3-month functional outcomes and using this definition there would be treatment benefit of alteplase over placebo at 24 hours. Methods- We analyzed the NINDS rt-PA Stroke Study (Parts 1 and 2) trial data. Percent change NIHSS was defined as ([admission NIHSS score-24-hour NIHSS score]×100/admission NIHSS score] and delta NIHSS as (admission NIHSS score-24-hour NIHSS score). We compared early neurological improvement using these definitions between alteplase versus placebo patients. We also used receiver operating characteristic curve to determine the predictive association of early neurological improvement with excellent 3-month functional outcomes (Barthel Index score of 95-100 and modified Rankin Scale score of 0-1), good 3-month functional outcome (modified Rankin Scale score of 0-2), and 3-month infarct volume. Results- There was a significantly greater improvement in the 24-hour median percent change NIHSS among patients treated with alteplase compared with the placebo group (28% versus 15%; P=0.045) but not median delta NIHSS (3 versus 2; P=0.471). Receiver operating characteristic curve comparison showed that percent change NIHSS (ROCpercent) was better than delta NIHSS (ROCdelta) and admission NIHSS (ROCadmission) with regards to excellent 3-month Barthel Index (ROCpercent, 0.83; ROCdelta, 0.76; ROCadmission, 0.75), excellent 3-month modified Rankin Scale (ROCpercent, 0.83; ROCdelta, 0.74; ROCadmission, 0.78), and good 3-month modified Rankin Scale (ROCpercent, 0.83; ROCdelta, 0.76; ROCadmission, 0.78). Conclusions- In the NINDS rt-PA trial, alteplase was associated with a significant percent change improvement in NIHSS at 24 hours. Percent change in NIHSS may be a better surrogate marker of thrombolytic activity and 3-month outcomes.

Are Stroke Survivors Discharged to the Recommended Postacute Setting?

OBJECTIVE: To examine the processes and barriers involved in providing postdischarge stroke care. DESIGN: Prospective study of discharge planners' (DP) and physical therapists' (PT) interpretation of factors contributing to patients' discharge destination. SETTING: Twenty-three hospitals in the northeastern United States. PARTICIPANTS: After exclusions, data on patients (N=427) hospitalized with a primary diagnosis of stroke between May 2015 and November 2016 were examined. Of the patients, 45% were women, and the median age was 71 years. DPs and PTs caring for these patients were queried regarding the selection of discharge destination. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Comparison of actual discharge destination for stroke patients with the destinations recommended by their DPs and PTs. RESULTS: In total, 184 patients (43.1%) were discharged home, 146 (34.2%) to an inpatient rehabilitation facility, 94 (22.0%) to a skilled nursing facility, and 3 (0.7%) to a long-term acute care hospital. DPs and PTs agreed on the recommended discharge destination in 355 (83.1%) cases. The actual discharge destination matched the DP and PT recommended discharge destination in 92.5% of these cases. In 23 cases (6.5%), the patient was discharged to a less intensive setting than recommended by both respondents. In 4 cases (1.1%), the patient was discharged to a more intensive level of care. In 2 cases (0.6%), the patient was discharged to a long-term acute care hospital rather than an inpatient rehabilitation facility as recommended. Patient or family preference was cited by at least 1 respondent for the discrepancy in discharge destination for 13 patients (3.1%); insurance barriers were cited for 9 patients (2.3%). CONCLUSIONS: Most stroke survivors in the northeast United States are discharged to the recommended postacute care destination based on the consensus of DP and PT opinions. Further research is needed to guide postacute care service selection.

Prevalence of a Multiple Territory Stroke Pattern After Intravenous Thrombolysis.

BACKGROUND: It has been proposed that the presence of a multiple territory stroke pattern (MTSP) on brain imaging may aid identification of patients with covert atrial fibrillation (AF). However, it is uncertain whether this association holds true among patients treated with intravenous recombinant tissue plasminogen activator (rtPA) because clot fragmentation may affect MTSP prevalence. METHODS/DESIGN: Retrospective analysis of 149 acute ischemic stroke patients treated with intravenous rtPA who underwent brain MRI. Presence of multiple acute infarctions on brain MRI that involved more than one vascular territory was considered to denote MTSP. Stroke etiology was categorized as nonembolic, cardioembolic (CES), and embolic stroke of undetermined source (ESUS). RESULTS: In the entire cohort, subjects with CES and ESUS had significantly more often an MTSP than subjects with other determined stroke mechanism (P= .007). Although numerically relatively more patients had an MTSP as compared to a non-MTSP among subjects with CES (52% versus 33.9%) and ESUS (44% versus 34.7%), this difference did not reach significance after Bonferroni-adjustment for multiple comparisons (P> .05, each). There was no difference in the prevalence of an MTSP among subjects with known (n = 11/51; 21.6%) versus subsequently diagnosed (n = 1/3; 33.3%) AF (P= .54). CONCLUSIONS: Our findings indicate that the known association of multiterritory infarct with AF and ESUS is maintained after thrombolysis. In light of its high specificity, MTSP represents a good marker for AF-related stroke etiology; nevertheless, overall sensitivity for AF was low highlighting that an absent MTSP does not rule out AF.

COVID-19 Pandemic and Burden of Non-Communicable Diseases: An Ecological Study on Data of 185 Countries.

BACKGROUND: The interaction between coronavirus disease 2019 (COVID-19) and non-communicable diseases may increase the global burden of disease. We assessed the association of COVID-19 with ageing and non-communicable diseases. METHODS: We extracted data regarding non-communicable disease, particularly cardiovascular disease, deaths, disability-adjusted life years (DALYs), and healthy life expectancy (HALE) from the Global Burden of Disease Study (GBD) 2017. We obtained data of confirmed COVID-19 cases, deaths, and tests from the Our World in Data database as of May 28, 2020. Potential confounders of pandemic outcomes analyzed include institutional lockdown delay, hemispheric geographical location, and number of tourists. We compared all countries according to GBD classification and World Bank income level. We assessed the correlation between independent variables associated with COVID-19 caseload and mortality using Spearman's rank correlation and adjusted mixed model analysis. FINDINGS: High-income had the highest, and the Southeast Asia, East Asia, and Oceania region had the least cases per million population (3050.60 vs. 63.86). Sub-saharan region has reported the lowest number of COVID-19 mortality (1.9). Median delay to lockdown initiation varied from one day following the first case in Latin America and Caribbean region, to 34 days in Southeast Asia, East Asia, and Oceania. Globally, non-communicable disease DALYs were correlated with COVID-19 cases (r = 0.32, p<0.001) and deaths (r = 0.37, p<0.001). HALE correlated with COVID-19 cases (r = 0.63, p<0.001) and deaths (r = 0.61, p<0.001). HALE was independently associated with COVID-19 case rate and the number of tourists was associated with COVID-19 mortality in the adjusted model. INTERPRETATION: Preventive measures against COVID-19 should protect the public from the dual burden of communicable and non-communicable diseases, particularly in the elderly. In addition to active COVID-19 surveillance, policymakers should utilize this evidence as a guide for prevention and coordination of health services. This model is timely, as many countries have begun to reduce social isolation.