Is the COVID-19 pandemic and stressor exposure associated with caregivers’ perceptions of children’s psychological well-being?In this survey study among 32 217 caregivers of public-school students, endorsement of child mental health concerns was significantly higher and endorsement of positive adjustment characteristics was significantly lower after the end of in-person instruction compared with before. After accounting for covariates, child mental health concerns increased in probability and positive adjustment characteristics decreased in probability as COVID-19 exposure and family stressors increased.These findings suggest that COVID-19 was associated with negative caregiver perceptions of children’s psychological well-being, requiring a comprehensive public health strategy. This survey study examines the associations of school closure and exposure to COVID-19–related stressors with caregivers’ perceptions of their children’s mental well-being. Understanding youth well-being during the COVID-19 pandemic can help appropriately allocate resources and inform policies to support youth.To examine caregiver-reported changes in the psychological well-being of their children 3 to 4 months after the start of COVID-19 stay-at-home orders, and to examine the association of caregiver-reported COVID-19 exposure and family stressors with caregiver perceptions of child psychological well-being.This survey study used an anonymous survey distributed via email from June 24 to July 15, 2020, to 350 000 families of students attending public schools in Chicago, Illinois. The a priori hypotheses were that caregivers would report worsening in child psychological well-being during the closure period compared with preclosure and that exposure to COVID-19–related stressors would be associated with a higher probability of worsening child psychological well-being. Data were analyzed from September 10, 2020, to March 15, 2021.Outcomes were 7 mental health concerns and 5 positive adjustment characteristics reported by caregivers using a retrospective pre-post design. COVID-19 exposure and family stressors were also reported by caregivers.P < .001) to 28.3 percentage points  higher after the end of in-person instruction compared with preclosure. Frequency of caregiver endorsement of youth positive adjustment characteristics ranged from −13.4 percentage points  to −30.9 percentage points  lower after the end of in-person instruction. Significant differences in COVID-19 exposure were observed across racial/ethnic  and household income strata . After accounting for covariates, all mental health concerns increased in probability  and all the positive adjustment characteristics decreased in probability  as COVID-19 exposure and family stressors increased.Among 350 000 families invited to participate, 32 217 caregivers  completed the survey on behalf of 49 397 children in prekindergarten through 12th grade. Child-specific outcomes were reported for 40 723 to 40 852 children depending on the specific question. The frequency of caregiver endorsement of youth mental health concerns ranged from 0.1 percentage point (suicidal ideation or self-harm, reported by 191 caregivers [0.5%] preclosure vs 246 caregivers [0.6%] during closure; In this survey study of caregivers during the COVID-19 pandemic, COVID-19 and resulting exposure to stress were associated with worse youth psychological well-being, demonstrating the need for a comprehensive public health approach that prioritizes children’s well-being and draws broad public attention to the mental health needs of youth. Of note, the 10% of families in this sample who reported simultaneous worsening in parental mental health and children’s behavioral health indicated high rates of exposure to COVID-19–related stressors, such as the loss of childcare, food insecurity, and disruption in health care. A study by Gassman-Pines et al13 of hourly service workers with young children showed that even in the first weeks after implementation of pandemic restrictions, families experiencing more COVID-19–related financial stressors reported worsening psychological well-being for themselves as well as their children.Despite these concerns, few studies have examined youth mental health during the COVID-19 pandemic. Studies from early in the pandemic show an increase in depressive and anxiety symptoms among adolescents.14 In contrast, significant increases in children’s self-reported symptoms of depression, self-harm, suicidal ideation, plans, and attempts were found immediately after school-reopening in China compared with immediately prior to the outbreak of the pandemic.15 An analysis by Leeb et al16 reported that although the overall number of mental health–related emergency department visits declined from March to May of 2020, the overall proportion of emergency department visits related to mental health concerns increased by 24% for children and by 31% for adolescents from March to October 2020 compared with the same time period in 2019.Less is known about risk for serious mental health concerns during the pandemic, such as suicide. In the first 3 months of school closures in Japan, no differences in suicide rates for youth younger than age 20 years were found compared with the same timeframe in 2018 and 2019.This survey study used survey data from a large sample of caregivers whose children attended public school in Chicago, Illinois, to examine 2 hypotheses: that caregivers would report worsening in the psychological well-being of their children 3 to 4 months after the start of stay-at-home orders and resulting school closures and that exposure to COVID-19–related stressors would be associated with a higher probability of caregiver-reported worsening in child psychological well-being.The research review board at the Chicago Public Schools and Northwestern University’s institutional review board approved all study activities and exempted the study from written and oral consent because the study was anonymous and low risk.The data for this study are from a cross-sectional survey examining caregiver attitudes about returning to in-person instruction after remote learning during the first phase of the COVID-19 pandemic in the United States. The survey was developed by a multidisciplinary team. Expert panel review was conducted, and the draft survey was pretested to ensure clarity, validity, and reliable functioning of all questions and response options before dissemination. Individuals were eligible to participate if they were a caregiver of a current student in the school district and were aged at least 18 years. For recruitment, the school district publicly distributed an email invitation and survey link to families that represent 350 000 students. The survey was offered in English, Polish, Spanish, and Chinese, the 4 most widely spoken languages among families in the school district.The school district identified a subset of 171 127 students from 365 schools with a less than 5% response rate or without a parent email on file. From this subset, the research team attempted telephone outreach to 179 additional families. Of these, 22 families completed the survey via telephone. Families who did not participate did not have working telephone numbers (59 families), declined to take the survey (38 families), were left voicemails (29 families), or were otherwise ineligible to participate  (31 families).Caregivers were asked to complete the survey regarding 1 of their children in the school system with the option to complete the survey for additional children. To document disparities shaped by structural racism, caregivers were asked to self-report on race/ethnicity using preset options or indicate they preferred not to answer. The survey remained open for responses from June 24, 2020, to July 15, 2020. The deidentified data set was shared with the research team by the school district for analyses.17 We omitted 5 items because nearly all participants were expected to respond affirmatively . A total CEFIS was derived by summing the number of specific CEFIS indicators selected by participants with complete data on all 20 items.The survey included 20 out of 25 items from the COVID-19 Exposure and Family Impact Scale (CEFIS).18 Parents were asked, “Which of the following generally describe your child BEFORE the end of in-person instruction on March 17, 2020?” Response options included 7 mental health concerns  and 5 positive adjustment characteristics . A follow-up question asked caregivers to indicate “Which of the following generally describe your child SINCE the end of in-person instruction on March 17, 2020?” Response options included the same 7 mental health concerns and 5 positive adjustment characteristics. Respondents were instructed to select all that applied to their child. Owing to low base rates, items assessing self-harm and thoughts of suicide were combined for analyses.Items assessing psychological well-being were developed specifically for this survey using a retrospective pre-post design.lrm command from the rms R package to better understand associations between CEFIS score and caregiver-reported changes in specific mental health concerns and positive adjustment characteristics in the child. Caregiver-reported presence or absence of each characteristic since the end of in-person instruction  was modeled as the outcome. Caregiver-reported presence or absence of each specific characteristic preclosure was included as a covariate, along with city region . Given racial/ethnic and income disparities in exposure to COVID-19,19 race/ethnicity and household income were also included as covariates. Cluster robust SEs were specified at the household level to account for clustering of multiple children per household using the robcov function, which implements the Huber-White method. Marginal effects at the mean of each covariate adjusted model were derived and visualized via ggpredict, from the ggeffects package in R.Summary descriptive statistics were calculated using R statistical software version 4.0 , which included raw counts and percentages for categorical variables, and means with SDs for CEFIS scores. Pairwise deletion of individuals with missing data on specific variables was used for all other analyses. This strategy leverages all available data and does not entirely omit individuals with missing data for some, but not all, variables of interest. One-way analysis of variance was used to test whether continuous CEFIS scores were significantly different across racial/ethnic and household income strata. A series of multiple logistic regression models were fit with the P values were 2-sided, and statistical significance was set at P = .05. Data were analyzed from September 10, 2020, to March 15, 2021.Surveys were completed by a racially/ethnically diverse sample of 32 217 caregivers, including 10 827 White caregivers (39.3%), 8320 Latinx caregivers (30.2%), 6168 Black caregivers (22.4%), and 2223 caregivers (8.1%) with multiple or other races/ethnicities . ApproxiP < .001) to 28.3 percentage points higher  (P < .001) to −30.9 percentage points lower   . In cont < .001) . PearsonBetween 5236 youths (12.8%) and 12 351 youths (30.2%) were described by caregivers as angry, anxious, depressed, lonely, or stressed during closure, but not preclosure  and household income strata . Across the entire sample, respondents reported a mean (SD) CEFIS score of 2.5 (2.2). Mean (SD) CEFIS scores were higher among Black (2.8 [2.4]) and Latinx (3.1 [2.3]) respondents compared with non-Latinx White respondents (1.8 [1.8]) (8 [1.8]) . Signifi8 [1.8]) .P < .001) and all the positive adjustment characteristics significantly decreased in probability  as CEFIS score increased (eTable 3 in the 20 and disrupt caregivers’ ability to regulate their own emotions and manage children’s behaviors.21 This transactional pattern has been associated with coercive parenting practices that may exacerbate children’s behavior problems22 and increase risk for child maltreatment.23 Increased loneliness may impact developing social skills among younger children and increase adverse health outcomes, poor academic performance, anxiety, depression, and suicide among adolescents.24In this survey study, caregivers reported pervasive negative perceptions of their children’s psychological well-being associated with the COVID-19 pandemic. After the end of in-person instruction, nearly a quarter of children were described by their caregivers as angry or agitated, more than one-quarter of children were described by their caregivers as anxious and stressed, more than one-third were described as lonely, and fewer than one-third of caregivers reported that their child had positive peer relationships. Children’s anger and behavioral challenges influence caregiving stress25 estimates indicate that 18.8% of students seriously considered suicide and 8.9% attempted suicide in the previous year. Given that youth suicide rates have increased by nearly 60% over the past 10 years,26 additional research is needed to better understand the impact of COVID-19 on suicidal ideation and behavior.Rates of caregiver endorsement of children’s self-harm behaviors and suicidal ideation were low prior to and after school closure was enacted, with only a slight increase after closure. These findings should be interpreted with caution, as caregivers may be unaware of their children’s self-harming behavior or suicidal ideation. When surveyed directly, Centers for Disease Control and PreventionThe findings of this survey study suggest that the COVID-19 pandemic is associated with widespread effects on children’s mental health, linked not only to children’s mood and peer relationships but also to their positive adjustment and familial interactions. For all youth, COVID-19 was associated with prolonged disruptions in daily routines, decreased exposure to adult and peer relationships, and reduced opportunities for academic and social development. Compounding these issues are the economic hardship, grief and loss, and health concerns that some youth have experienced as a result of COVID-19. In our sample, Black, Latinx, and youth living in poorer households were most likely to experience these additional stressors. Only 3 of 20 items on the CEFIS showed no significant differences by race/ethnicity, likely owing to the nearly universal consequences of widespread stay-at-home orders early in the pandemic. Given that non-Latinx White and wealthier households were more likely to report mental health concerns and less likely to report positive adjustment characteristics during closure, future research should examine differences in the processes of resilience among racially and ethnically marginalized households.27 Such approaches must include primary prevention efforts that target known risk factors likely to be caused or exacerbated by COVID-19, such as poverty, socioeconomic inequalities, health disparities, and stress. As our findings demonstrate, Black, Latinx, and low-income families reported disproportionately high rates of COVID-19 stressor exposure, likely attributable to systemic racism and ensuing structural inequities, such as underresourcing and lack of access to health care.7Our findings indicate a need for a research-informed public health approach that prioritizes children’s well-being and mental health.1 In addition, engaging youth and community members in policy and program development is critical to ensure that mental health promotion efforts are contextually and culturally relevant.28 Needs of communities vary widely, and funds should be directed and allocated in ways that will have the largest and most meaningful impact while not exacerbating existing inequities.Primary prevention efforts must be paired with targeted assessments to identify youth in need of support and policies that support equitable access to mental health care. New approaches are needed, such as brief and low-intensity interventions that can be implemented by individuals without specialized training.31 Schools have the potential to serve as hubs that support community healing and align public health strategies across child-serving systems. However, the public education system, already burdened by the need to adapt traditional models of education to the constraints of public health guidelines, will need additional support to respond in innovative ways to the increased mental health needs. There will also be a need to shore up access to school- and community-based behavioral health services, including tele–behavioral health services. Resources must also be provided to support educator health and well-being to address workplace stress and secondary traumatic stress among teachers.32 Finally, social justice requires that policy efforts are crafted to unmask, disrupt, and address disparities in COVID-19 needs and resources.33Prior to the pandemic, schools represented a major access point for food, health care, and mental health services.34 This response bias may lead to underestimates of the effects on psychological well-being, as it is possible that nonrespondents were experiencing higher levels of COVID-19–related stress than respondents. It is imperative that future research better engage racially marginalized families, low-income families, and immigrant and non-English speaking families to understand the true mental health impact of COVID-19 stressors. Second, the data rely exclusively on caregiver report. It has long been noted that cross-informant correlations on mental health concerns tend to be low to moderate,35 underscoring the importance of obtaining self-reports for youth. Third, this study used single-item, face valid questions to assess mental health concerns and positive adjustment rather than normed measures of mental health problems or adaptive functioning, limiting understanding of clinically significant distress or impairment. Fourth, school district leadership determined that the survey should be administered as rapidly as possible within a limited time to inform school district policy around school reopening. This necessitated the email distribution of deidentified survey links in lieu of individualized email invitations, thereby preventing the derivation of true survey response rates and raising the possibility—albeit unlikely—of multiple responses per household. The short survey field period also contributed to the very low rate of telephone follow-up. Additionally, the data were collected at a single point in time and rely on retrospective recall. As the COVID-19 pandemic continues, longitudinal data will help clarify whether initial distress is reduced, maintained, or intensified over time as well as identify subgroups of youth most at risk for continued concerns.This study has several limitations. First, data were collected from a sample of families residing in a large urban area and may not be representative of rural and suburban areas or areas with different COVID-19 exposure, social distancing policies, and resources. Furthermore, although the sample was sociodemographically diverse, it was not representative of families in the district. Based on publicly available district data, a higher percentage of survey respondents were White, English-speaking, and had higher income levels compared with district demographic characteristics.The findings of this survey study suggest that COVID-19 and resulting exposure to stress were associated with negative caregiver perceptions of children’s psychological well-being. The prevalence of these concerns demonstrate the need for a comprehensive public health approach that prioritizes children’s well-being and draws broad public attention to the mental health needs of youth.


Background: To date, the role of bridging intravenous thrombolysis before mechanical thrombectomy (MTE) is controversial but still recommended in eligible patients. Different doses of intravenous alteplase have been used for treating patients with acute ischemic stroke from large-vessel occlusion (LVO-AIS) in Asia, largely due to variations in the risks for intracerebral hemorrhage (ICH) and treatment affordability. Uncertainty exists over the potential benefits of treating low-dose alteplase, as opposed to standard-dose alteplase, prior to MTE among patients with LVO-AIS.Aim: The aim of the study was to compare outcomes of low- vs. standard-dose of bridging intravenous alteplase before MTE among LVO-AIS patients.Methods: We performed a retrospective analysis of LVO-AIS patients who were treated with either 0.6 mg/kg or 0.9 mg/kg alteplase prior to MTE at a stroke center in Northern Vietnam. Multivariable logistic regression models, accounting for potential confounding factors including comorbidities and clinical factors , were used to compare the outcomes between the two groups. Our primary outcome was functional independence at 90 days following stroke . Secondary outcomes included any ICH incidence, early neurological improvement, recanalization rate, and 90-day mortality.Results: We analyzed data of 107 patients receiving bridging therapy, including 73 with low-dose and 34 with standard-dose alteplase before MTE. There were no statistically significant differences between the two groups in functional independence at 90 days  after accounting for potential confounding factors. Compared to the standard-dose group, patients with low-dose alteplase before MTE had similar rates of successful recanalization, early neurological improvement, 90-day mortality, and ICH complications.Conclusion: In the present study, patients with low-dose alteplase before MTE were found to achieve comparable clinical outcomes compared to those receiving standard-dose alteplase bridging with MTE. The findings suggest potential benefits of low-dose alteplase in bridging therapy for Asian populations, but this needs to be confirmed by further clinical trials. The role of bridging thrombolysis before mechanical thrombectomy (MTE) has been controversial , but it In 2010, an open-label, nonrandomized, observational study suggested that low-dose 0.6 mg/kg) intravenous alteplase within 3 h of stroke onset could be safe and effective for the Japanese population . Finding mg/kg inThe aim of the study was to compare clinical outcomes of low-dose vs. standard-dose intravenous alteplase combined with MTE in patients with LVO-AIS.The study was conducted at the Stroke Center of Bach Mai Hospital, Hanoi, Vietnam. As one of the leading stroke centers nationally, we have been providing acute care to approximately 10,000 episodes of stroke each year. We retrospectively included patients with LVO-AIS at the Stroke center who received bridging therapy between 2017 and 2019. The included patients must meet the criteria for treatment of intravenous alteplase within a 4.5-h window from stroke onset as well as MTE within the 6-h window from stroke onset. We applied the inclusion and exclusion criteria for intravenous alteplase within 4.5 h of onset as recommended in the 2013 American Heart Association/American Stroke Association (AHA/ASA) Guidelines ] and theThe Japanese drug safety authority has approved the use of low-dose alteplase after an open-label, nonrandomized, observational study showed that it could be safe and effective compared to standard-dose alteplase for the Japanese population . Given tAll the included patients received computed tomography (CT) and computed tomography angiography (CTA) scans to decide if alteplase should be administered. The patients were immediately referred to the intervention room for MTE if the CTA revealed an anterior large artery occlusion. MTE was performed via a transfemoral approach with either SOLITAIRE (stent retriever system), PENUMBRA (aspiration system), or SOLUMBRA (aspiration plus extraction technique).This retrospective study was approved by the Ethics Committee of Hanoi Medical University, No 187/HĐĐĐĐHYHN on February 20, 2016.Patients were assessed by clinicians and their data were obtained at 24, 72 h, 7 days (or at discharge if sooner), 28, and 90 days after hospital admission.Clinical assessments were performed using the National Institute of Health Stroke Scale (NIHSS), modified Rankin scale (mRS), and brain imaging CT scan, and/ or magnetic resonance imaging [MRI] within 24 h of admission. Information on patient characteristics and care received in the hospital was obtained from the medical records. They included (1) socio-demographics: age, sex; (2) pre-stroke factors: pre-morbid mRS, use of medications, risk factors , and comorbidities ; and (3) stroke-related clinical factors. The clinical factors included stroke severity , ischemic stroke subtype according to the Acute Stroke Treatment (TOAST) criteria , onset tThe primary outcome was functional independence at 90 days (defined as mRS 0–2). Secondary outcomes included early neurological improvement, successful reperfusion following the MTE procedure, any ICH complications, and 90-day mortality. Early neurological improvement was defined as a reduction of at least eight points in the NIHSS score or an NIHSS score of 0 or 1 at 72 h after hospitalization . Recanalt-test or Mann–Whitney U-test, where relevant, for continuous variables. The Fisher exact test was applied for continuous variables when the number of observations was <5. A two-sided p-value < 0.05 was considered statistically significant. Multivariable logistic regression models, accounting for potential confounding factors , were used to compare clinical outcomes and hemorrhagic complications between the two groups  following bridging therapy. We also performed sensitivity analyses that included some relevant covariates not meeting the criteria for being confounders as specified above to see if these factors impacted different outcomes.All the data analyses were performed using SPSS software, version 21.0 . Parameters were presented as N (%), mean ± standard deviation, or median (25 and75th percentile). Differences between the two groups were assessed using the Pearson correlation test for categorical variables and the Student p < 0.001), and longer onset to hospital arrival time [mean difference (MD): 26.6 min; p = 0.005; p = 0.094) and 12 min  in the low-dose group (78.1%) than in the standard-dose group (26.5%), while the use of the solumbra technique was more common in the standard-dose than the low-dose group . However, there were no significant differences between the two groups in successful recanalization rates (mTICI score 2b−3) before thrombectomy  who were treated with intravenous alteplase prior to MTE. They were divided into two groups according to the bridging therapy they received: 73 were treated with a lower dose of intravenous alteplase (0.6 mg/kg) and MTE between 2017 and 2018, and 34 were treated with a standard dose of intravenous alteplase (0.9 mg/kg) and MTE in 2019. The factors that met our criteria for being included in the multivariable models were age, sex, history of hypertension, initial NIHSS, onset to needle time, needle to groin time, and where applicable, the MTE technique was used. After accounting for potential confounding factors , it was N = 42; Korea; N = 64; the present study; N = 107).Our single-center retrospective study showed no statistically significant differences in clinical outcomes and ICH complications between two groups of LVO-AIS patients treated with low-dose vs. standard-dose bridging alteplase before MTE, after accounting for confounding factors. The findings are consistent with those from the Korean and TaiwaFor both groups, MTE was performed immediately following thrombolysis and we continued alteplase infusion during thrombectomy, even with those who achieved successful recanalization. High recanalization rates (>95%) presented in this study are consistent with previous studies conducted in Asian populations. In the SKIP trial conducted in Japan, the rate of successful reperfusion was 92% among those receiving low-dose alteplase bridging with MTE . SimilarOur study has several limitations and strengths. This is a single-center retrospective study that included patients receiving either standard- or low-dose alteplase prior to MTE from two different time periods. There could be a risk of selection bias due to the lack of randomization. We attempted to account for the differences in patient characteristics and clinical factors between two groups in our data analyses. However, residual confounding is likely due to poorly measured  or unmeasured confounding factors  in the study. The sample size was small, which limited our study power particularly for comparing secondary outcomes  among the two groups of patients. We were also unable to perform sensitivity analyses using alternative statistical methods, such as the propensity score matching approach. To our knowledge, this is one of the rare three studies , 19, allThe study findings show no differences in clinical outcomes and ICH complications between low-dose vs. standard-dose bridging alteplase before MTE. The number of treated patients was too low to show non-inferiority of low-dose treatment. The approach of treating low-dose alteplase patients with LVO-AIS may prove beneficial by future studies for cases presenting directly to a thrombectomy-capable stroke center and to Asian populations with higher bleeding risks. Larger randomized trials are needed to demonstrate whether a low alteplase dose retains the same efficacy as the standard dose. The role of tenecteplase (TNK) in bridging therapy could also prove to improve clinical outcomes in patients requiring bridging therapy. According to the Tenecteplase vs. Alteplase before Endovascular Therapy for Ischemic Stroke (EXTEND-IA TNK) trial , TNK befThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This retrospective study was approved by the Ethics Committee of Hanoi Medical University, No 187/HÐÐÐÐHYHN on February 20, 2016.All authors made substantial contributions to the study concept and design, acquisition of data, or data analysis, and interpretation of data. The authors also took part in drafting the article, and/or revising the manuscript critically for important intellectual content, read and gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: This study aimed to identify maternal circulating exosomal miRNAs as potential non-invasive biomarkers for the early detection of fetal ventricular septal defects (VSDs).Methods: In total, 182 pregnant women, comprising 91 VSD cases and 91 matched controls, were included in this study. Exosomes were isolated; dysregulated exosomal miRNAs were profiled using next-generation sequencing. Differential abundance of miRNAs was verified using quantitative real-time polymerase chain reaction (qRT-PCR). Diagnostic accuracy was evaluated by constructing receiver operating characteristic (ROC) curves.Results: In total, 77 serum exosomal miRNAs were found to be differentially expressed in the VSD group compared to their expression in the control group. Among these, five downregulated exosomal miRNAs were validated using qRT-PCR. hsa-miR-146a-5p was identified to be capable of distinguishing VSD cases from controls .Conclusion: Circulating exosomal miRNAs, particularly hsa-miR-146a-5p, may be predictive biomarkers for the non-invasive prenatal diagnosis of fetal VSDs. Congenital heart disease (CHD) is the most common type of birth defect and has an incidence of nearly 8 of every 1,000 live births worldwide . CHD repCurrently, the diagnosis of CHD is mainly dependent on fetal echocardiography during 22–28 weeks of pregnancy. However, CHD diagnosis with the use of echocardiography is partially limited by the skill of the operator, quality of equipment, and lack of process standardization. Therefore, there is an urgent need to identify early biomarkers in the maternal blood to enable accurate screening of fetal CHDs for more sensitive prenatal diagnosis. Recently, exosomal miRNAs have attracted considerable attention as disease biomarkers. In this study, we focused on identifying biomarkers of fetal VSDs by profiling maternal serum exosomal miRNAs.Exosomes are extracellular vesicles (EVs) that contain macromolecules such as proteins, miRNAs, and mRNAs that can modulate target cell biology and function . MultiplAlthough exosomal miRNAs in maternal circulation have gained much interest for the detection of pregnancy-associated disorders, few studies have focused on the connection between exosomal miRNAs and fetal CHDs. We hypothesized that maternal serum exosomal miRNAs are potential candidate biomarkers for early prenatal detection of fetal VSDs.In our study, we investigated miRNAs in EVs isolated from maternal serum as potential non-invasive screening biomarkers for fetal VSD. We analyzed exosomal miRNA profiles of maternal serum from pregnant women carrying fetuses with VSD as well as from pregnant women carrying normal fetuses as a control. The study identified hsa-miR-146a-5p as a potential novel early detection marker for VSD.The study was conducted on a retrospective cohort of pregnant women at 16–18 weeks of gestation who were admitted to the prenatal diagnosis center of Zhejiang Jiaxing Maternal and Child Health Care Hospital between January 2017 and January 2020. The cohort consisted of 91 pregnant women carrying fetuses with isolated VSD and 91 women carrying normal fetuses, as identified by prenatal cardiac ultrasound scanning. The disease and control groups were matched by maternal age, gestational age, and sampling date. All participants remained healthy and had no family genetic history. The clinical characteristics of the subjects are summarized in The study was divided into two stages. In the first stage (biomarker discovery), we created nine pools of serum samples from 36 VSD samples and 36 control samples, with each pool consisting of four samples. We then identified the differential miRNA profiles of the 2 groups by sequencing. In the second stage , we conducted quantitative reverse transcription polymerase chain reaction (qRT-PCR) on samples from the remaining 55 pregnant women carrying fetuses with VSD and 55 women carrying normal fetuses to validate the miRNAs that were identified in the first stage. A receiver operating characteristic (ROC) curve was used to assess the accuracy of maternal serum miRNA in predicting fetal VSD.g for 10 min and at 16,000 × g for 10 min) to remove cell debris and blood platelets. The supernatant was transferred to Eppendorf tubes for exosome isolation, miRNA extraction, and miRNA sequencing. Circulating exosomes were isolated from serum using RiboTM Exosome Isolation Reagent according to the manufacturer’s instructions .Each 4-mL serum sample pool was centrifuged twice  were used to validate the miRNA sequencing results via real-time qRT-PCR.Serum exosome samples (20 μL) were transferred to a copper mesh. Next, the sample was stained and fixed with 2% uranyl acetate for 1–10 min. The mesh was placed on a filter paper and air-dried. The morphological features of the exosomes were examined and visualized using TEM  at a magnification of 44,000 × (200 nm).The exosome particle quantity, size, distribution, and concentration were measured using NTA at VivaCell Biosciences with ZetaView PMX 110  and the corresponding software ZetaView 8.04.02.Isolated exosome samples were appropriately diluted in 1 × phosphate-buffered saline (PBS), and NTA measurements were recorded and analyzed at 11 positions. The ZetaView system was calibrated using 110-nm polystyrene particles. The temperature was maintained at approximately 23–30°C.® West Dura Extended Duration Substrate according to the manufacturer’s instructions.Exosomal protein was dissolved in radioimmunoprecipitation assay lysis buffer, and the total protein concentration was determined by a BCA protein assay. Approximately 60 μg of protein was subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked for 1 h with 5% bovine serum albumin, and incubated with antibodies against CD63, CD9, and CD81(Abcam) at 4°C overnight, followed by incubation with a secondary antibody  at 4°C for 1 h. Specific proteins were detected using SuperSignal®2.0  and the Agilent 2200 TapeStation , respectively.Total RNA was extracted and purified from exosomes using the Liquid miRNA Kit/HiPure Serum/Plasma miRNA Kit . The quantity and integrity of exosomal RNA yield were assessed using Qubit® Multiplex Small RNA Library Prep Set for Illumina  according to the manufacturer’s instructions. Small RNAs were reverse transcribed and amplified by PCR. Next, PCR products were sequenced by HiSeq 2500  with a single-end 50-bp from Ribobio Co., Ltd. .Exosomal RNA (50 ng) from each sample pool was used to prepare small RNA libraries using the NEBNextp-value < 0.05 indicates a score representing significant GO-annotation enrichment for the differentially expressed genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze signaling pathways related to target genes. The p-value was determined by Fisher’s exact test.To predict the target genes of the differentially expressed miRNAs, data from TargetScan, miRDB, miRTarBase, and miRWalk were integrated into our proprietary database. Only overlapping results of the four databases were accepted as potential target genes. Gene ontology (GO) analysis provides meaningful gene and gene products and covers three domains: biological processes (BP), cellular component (CC), and molecular function (MF). A TM miRNA qRT-PCR Starter Kit  and miRNA-specific stem-loop primers were used to reverse transcribe total RNA. Based on the results of target gene prediction and enrichment analysis as well as literature, five exosomal miRNAs  were selected to validate the miRNA sequencing results using an independent cohort of 110 exosomal samples  via qRT-PCR. Extracted RNA was reverse transcribed into cDNA, which was subjected to real-time qRT-PCR using a Bulge-LoopTM miRNA qRT-PCR Starter Kit on a CFX Real-time PCR system . Cel-miR-39 was added as an external control and normalized for technical variation between the samples, as described previously  were used to assess the sensitivity and specificity of the exosomal miRNAs for prenatal diagnosis of fetal VSD. All statistical calculations were performed using SPSS 23.0 and GraphPad Prism 6.0.A two-tailed Student’s The total miRNA of the maternal serum exosomes from nine VSD sample pools and nine control sample pools was extracted for miRNA sequencing. The clinical characteristics of the 72 participants in the first stage and the other 110 participants in the second stage are shown in n = 5 per group) to evaluate the presence of the exosomal protein markers CD9, CD63, and CD81 through western blotting; the characteristics of the VSD and control samples were not significantly different ] are presented in The top 10 GO categories for dysregulated miRNAs ranked by enrichment score [−log10(p < 0.05) for targets of deregulated miRNAs. As shown in KEGG pathway analysis revealed 237 pathways that were significantly enriched  were shortlisted based on their fold changes and bioinformatics analysis as well as the literature associated with CHD  reads > 100; (2) | log2FoldChange| > 2; (3) with CHD . The utiwith CHD . HoweverHsa-miR-146a-5p possessed the highest diagnostic power in discriminating fetuses with VSD and healthy fetuses, with an AUC of 0.997 . The AUC of hsa-miR-199a-3p was 0.6717 . These results indicate that hsa-miR-146a-5p can distinguish cases of fetal VSD from controls with high accuracy and may serve as an efficient and non-invasive biomarker for the prenatal detection of fetal VSD .We retrospectively screened exosomal miRNAs from serum of pregnant women carrying fetuses with VSD or carrying normal fetuses at 16–18 weeks of gestation. In our study, we first analyzed the expression profile of exosomal miRNAs in maternal serum using sequencing technology, revealing 77 exosomal miRNAs that were differentially expressed in the serum of women carrying VSD fetuses compared to that in the serum of women carrying normal fetuses; of these 77 exosomal miRNAs, 12 were significantly upregulated and 65 downregulated in the VSD group. Following validation via qRT-PCR and ROC analysis, we demonstrated exosomal hsa-miR-146a-5p has high diagnostic performance in terms of sensitivity and specificity in differentiating cases of fetal VSDs from controls. These results imply that exosomal miRNAs may be useful predictive biomarkers for the non-invasive prenatal screening of fetal CHDs.Previously, some studies have identified miRNAs in maternal circulation to be non-invasive biomarkers for prenatal diagnosis of CHDs . HoweverGO category annotation and KEGG pathway enrichment analyses were performed to gain insight into the biological functions and molecular mechanisms of deregulated exosomal miRNAs in the evolution of VSDs. The top 10 biological processes identified in the present study include developmental processes, particularly the single-organism developmental process. This result indicates that dysregulated miRNAs might participate in organism development during VSD pathogenesis. In KEGG pathway analysis, some classical pathways, such as the PI3K-Akt, MAPK, cAMP, mTOR, and Hippo signaling pathways , have bePMAIP1, NUMB, ERBB4, IRAK1, and CCL5 are potential target genes of hsa-miR-146a-5p; these genes are closely related to cardiac morphogenesis and development in five signaling pathways, including the Notch and ErbB signaling pathways. Our study predicts that hsa-miR-146a-5p participates in the Notch signaling pathway and that the putative target gene NUMB is involved in cardiac morphogenesis. In mice, Numb functions redundantly and has been shown to be essential for epicardial development, cardiac progenitor cell differentiation, outflow tract alignment, atrioventricular septum morphogenesis, myocardial trabeculation, and compaction  or cardiosphere-derived cell (CDC) exosomes . MiR-146ath axes . Transfeath axes . Thus, eath axes , and recath axes . In thisHowever, this study has some limitations. First, although our study successfully identified exosomal hsa-miR-146a-5p as a promising VSD-related biomarker for prenatal diagnosis, the number of pregnant women carrying fetuses with VSD enrolled in the present study was limited. Additionally, the heterogeneity and genetic background of pregnant women could impact the accuracy of the diagnostic value of exosomal miRNAs. Further studies are necessary to assess these exosomal miRNA signatures in a larger independent cohort of patients and controls. Second, we focused on fetal VSD in this study, and in future research, we will elucidate the prognostic value of exosomal miRNAs isolated from maternal serum in the detection of the diverse subtypes of CHD. Third, all dysregulated exosomal miRNAs were identified in the maternal blood, and thus, we could not determine the exact origins of the diagnostic serum exosomal miRNA biomarker. Whether they are derived from the embryos, placenta, or mother requires further investigation.This study demonstrated the diagnostic value of exosomal hsa-miR-146a-5p in the serum of pregnant women carrying fetuses with, with excellent sensitivity and specificity. Our findings highlight the potential clinical utility of serum exosomal miRNAs as an alternative for the diagnosis of fetal CHDs. Further large-scale validation and blinded clinical trials are required to confirm the potential applicability of these markers in prenatal VSD diagnosis, and additional exploration and optimization are imperative to uncover the underlying molecular mechanism of exosomal hsa-miR-146a-5p in VSD pathogenesis.GSE176134.The original contributions presented in the study are publicly available in NCBI using accession number The studies involving human participants were reviewed and approved by the Ethics Committee of Women and Children’s Hospital Affiliated to Jiaxing University. The patients/participants provided their written informed consent to participate in this study.YJ and SL conceived the study, participated to its design and coordination, and wrote the manuscript. LA and YX carried out the assays and participated to designing the study. XC, LY, and YH carried out laboratory tests. PT and WZ prepared the figures and tables. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Statins may reduce a cytokine storm, which has been hypothesized as a possible mechanism of severe COVID-19 pneumonia. The aim of this study was to conduct a systematic review and meta-analysis to report on adverse outcomes among COVID-19 patients by statin usage.Literatures were searched from January 2019 to December 2020 to identify studies that reported the association between statin usage and adverse outcomes, including mortality, ICU admissions, and mechanical ventilation. Studies were meta-analyzed for mortality by the subgroups of ICU status and statin usage before and after COVID-19 hospitalization. Studies reporting an odds ratio (OR) and hazard ratio (HR) were analyzed separately.Thirteen cohorts, reporting on 110,078 patients, were included in this meta-analysis. Individuals who used statins before their COVID-19 hospitalization showed a similar risk of mortality, compared to those who did not use statins . Patients who were administered statins after their COVID-19 diagnosis were at a lower risk of mortality . The use of statins did not reduce the mortality of COVID-19 patients admitted to the ICU . Among non-ICU patients, statin users were at a lower risk of mortality relative to non-statin users .Patients administered statins after COVID-19 diagnosis or non-ICU admitted patients were at lower risk of mortality relative to non-statin users. In December 2019, the first outbreak of a novel human coronavirus infection named severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China [The pathophysiology that underlies the severe COVID-19 pneumonia is an overproduction of early response proinflammatory cytokines, namely tumour necrosis factor (TNF), IL-6 and IL-1β . If leftet al. [et al. [et al. [et al. [Several studies have reported on the use of statins amongst COVID-19 patients with differing conclusions. Zhang et al.  reported [et al.  reported [et al.  reportedet al. [et al. [Meta-analyses by Hariyanto et al.  and Kow  [et al.  have stuThe aim of this study was to conduct a systematic review and meta-analysis to report on adverse outcomes among COVID-19 patients by statin usage.Ovid MEDLINE, Embase and the Cochrane Central Register of Controlled Trials were searched from January 2019 to December 2020, for English-language publications. The search strategy is presented in Studies underwent level 1 title and abstract screening to identify articles that reported on the use of statins among COVID-19 patients. Relevant articles from level 1 screening subsequently underwent level 2 full-text screening. This step identified studies that reported on a clinical database for which adverse outcomes were reported. Reference lists of articles undergoing level 2 full-text screening were also assessed (i.e. backward reference searching), to identify other potentially relevant studies.All screening was done independently by two reviewers . Where there were disagreements, a discussion occurred and consensus was achieved to determine whether to include or exclude an article. Where consensus could not be achieved, a third author (NC) was consulted for their input and final decision.Articles identified for inclusion after level 2 full-text screening were assessed for quantitative synthesis. Articles were included if they reported an adjusted relative risk measure on mortality, ICU admissions or mechanical ventilation of patients who were statin users compared to those who were not statin users (comparison group).For studies included at this stage, demographics such as sample size, study design, patient population, central measures of tendency for age, percentage male, and percentage of statin users were noted. We noted whether studies reported exclusively on ICU patients at the time of enrollment. Additionally, we assessed if statin use was defined as prior to or after hospitalization.In corollary to study screening, quantitative synthesis was conducted independently by two reviewers , and a third reviewer (NC) was consulted if consensus was not achieved.2 of equal to or greater than 50%, a random-effects DerSimonian-Laird analysis model was applied; otherwise, a fixed-effects inverse-variance analysis model was used. A meta-regression was used, to assess the effect modifier of average age and sex of study participants. A p-value of less than 0.05 was considered statistically significant.Meta-analysis was conducted for mortality by the subgroups of ICU status  and statin usage . Studies employing similar relative risk ratios were analyzed together to generate a summary risk estimate. That is, studies reporting an odds ratio (OR) were aggregated to produce a summary odds ratio and studies reporting a hazard ratio (HR) were analyzed together. Corresponding 95% confidence intervals (CI) for an OR or a HR are reported as well. When there was heterogeneity of Ip-value of greater than 0.05 indicated a lack of asymmetry of the funnel plot, and thereby no significant concern for publication bias. All analyses were conducted using Stata 16.A funnel plot and Egger’s test were used to assess the publication bias for the endpoint of mortality. A A total of 482 records underwent level 1 screening, of which 474 were identified through database search and 8 identified from backward reference searching. After removing duplicates, 346 records underwent level 1 screening; 49 articles subsequently underwent level 2 screening. Twenty-one studies were assessed for quantitative synthesis, of which 13 studies , 13–22, et al. [et al. [et al. [Study demographics are presented in et al.  split th [et al.  and Rodr [et al. , reporte [et al. , 21, 22  [et al. , 20, 21  [et al. , 19, 22 p = 0.086) suggests no significant concern for publication bias  and OR of 0.62 . COVID-19 patients who had statins administered after their COVID-19 hospitalization had a lower risk of mortality compared to those who did not receive statins—HR of 0.53  and OR of 0.57  .COVID-19 patients who were using statins and admitted to the ICU did not show a lower risk of mortality, compared to non-statin users—OR of 0.65 . Among non-ICU patients, statin users were at a lower risk of mortality relative to non-statin users—HR of 0.53  and OR of 0.64  . Age waset al.–OR of 1.78 . It was also similar between patients who used and did not use statins after COVID-19 hospitalization, according to Song et al.–OR of 0.90 .Two studies reported on ICU admissions by patients who did and did not use statins. The risk of ICU admissions was similar between patients who used statins before COVID-19 hospitalization and who did not use statins, according to Yan et al. reported the odds of mechanical ventilation to be 0.99 , when comparing statin to non-statin users.Cariou et al. [et al. [et al. [et al. [This is the first rigorously conducted systematic review and meta-analysis investigating the association between statin use and adverse outcomes among COVID-19 patients, meeting the methodological guidance for a high quality systematic review and meta-analysis as recommended by the MOOSE group . With thet al.  and Kow  [et al. . The ana [et al.  reported [et al.  reportedet al. [et al. [Furthermore, this review employs different statistical methodologies than the prior reviews. While Kow et al.  jointly  [et al.  paper inThe results suggest that individuals with and without history of statin use before their COVID-19 hospitalization experience a similar risk of mortality, ICU admissions, and use of mechanical ventilation. However, we urge caution around this simple dichotomy of studies and the corresponding interpretation. Depending on the time sequence of events, we hypothesize there are four possible scenarios of statin use within these included studies :Patients who used statins prior to COVID-19 hospitalization and who continue to use statins after hospitalizationPatients who used statins prior to COVID-19 hospitalizations and who cease to use of statins after hospitalizationPatients who have no history of statin use prior to COVID-19 hospitalization and start using statins after hospitalizationPatients who have no history of statin use prior to COVID-19 hospitalization and do not receive statins after hospitalizationAs statins are not currently a recommended medication for COVID-19 treatment and there is no randomized controlled trial in this setting, one could assume that Scenario 3 is unlikely to happen given its intent to solely treat COVID-19 with statin.In the event that Scenario 3 did occur, patients in Scenario 3 who only used statins after COVID-19 hospitalization may have acute cardiovascular indications for statin use after hospitalization, such as those who may develop acute myocardial injury, acute coronary syndrome or stroke. Our analysis by statin use before COVID-19 hospitalization therefore compares patients who used statins prior to COVID-19 hospitalization who may or may not continue using statins (Scenarios 1 and 2) after hospitalization, to patients who did not use statins prior to COVID-19 hospitalization who may or may not start statins after hospitalization (Scenarios 3 and 4). The data reporting on non-statin users prior to COVID-19 hospitalization would therefore include more patients with poorer outcomes (Scenario 3), and result in an overestimation of the possible protective association between statin use before hospitalization and adverse outcomes.et al. [Along a similar vein, the possible protective association of statins prior to COVID-19 hospitalization and adverse outcomes may also be underestimated. Masana et al.  reportedThis review also reports that patients who used statins after their COVID-19 hospitalization were at a lower risk of mortality and this protective association between statins use and mortality may be underestimated. Of the four aforementioned scenarios, our comparison of statin use after COVID-19 hospitalization may be summarized as a comparison of patients who used statins after COVID-19 hospitalization regardless of prior usage (Scenarios 1 and 3), relative to patients who did not use statins after COVID-19 hospitalization regardless of prior usage (Scenarios 2 and 4). Again, in the event that Scenario 3 manifested and thereby the arm of patients who used statins after COVID-19 hospitalization experience poorer outcomes, the protective association between statins use after COVID-19 hospitalization and mortality may be underestimated.et al. also reported that patients who continued on statins had significantly better outcomes, compare to patients who were not on statins before COVID-19 (p = 0.045). In fact, our results suggest that statin usage after COVID-19 hospitalization may have a protective association for mortality, and our analysis supports the recommendation by UpToDate® for “continuing statins in hospitalized patients with COVID-19 who are already taking them” [Masana ng them” . HoweverOur analysis also suggests that the risk of mortality among statin users compared to non-statin users differs, when focusing on subsets of ICU and non-ICU patients. Non-ICU patients seemed to experience benefit from statin use, while this beneficial association was not seen among patients admitted to the ICU. It is possible that statin has a more beneficial effect to decrease inflammation, early in the stage of illness and among less sick patients with COVID-19. However, there is a paucity of data with respect to ICU patients. Further investigation is required among ICU patients, given this limited statistical power to find association between statin and mortality, among ICU patients.Of final note, we found no effect modification by age or sex. The observed effects of statins does not change as a function of male or female, and old or younger.This study was not without limitations. Intrinsic to meta-analysis study designs, the strength of meta-analysis conclusions is limited to the strength of the input studies and underlying data. As all included studies are observational studies, randomized controlled trials are needed to confirm whether statin can be beneficial in patients with COVID-19 as COVID-19-specific treatment agent. Additionally, there is a general paucity of literature. While there are thirteen studies reporting on mortality, only two studied ICU patients. Only two studies reported on ICU admissions; only one study reported on mechanical ventilation. We need further research to address whether statin is beneficial in terms of preventing ICU admission or mechanical ventilation, which are surrogate markers of severe COVID-19 infection.In conclusion, patients administered statins after COVID-19 diagnosis were at lower risk of mortality. Ultimately, all included studies in this analysis were observational and predominantly retrospective in nature. Further prospective investigation is needed to assess whether statins may be an effective therapy in COVID-19 patients. Continued investigation is required to assess statin use among COVID-19 patients with respect to other outcomes, such as ICU admissions and mechanical ventilation.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.


Human visual cortex contains three scene-selective regions in the lateral, medial and ventral cortex, termed the occipital place area (OPA), medial place area (MPA) and parahippocampal place area (PPA). Using functional magnetic resonance imaging (fMRI), all three regions respond more strongly when viewing visual scenes compared with isolated objects or faces. To determine how these regions are functionally and causally connected, we applied transcranial magnetic stimulation to OPA and measured fMRI responses before and after stimulation, using a theta-burst paradigm (TBS). To test for stimulus category-selectivity, we presented a range of visual categories . To test for specificity of any effects to TBS of OPA we employed two control conditions: Sham, with no TBS stimulation, and an active TBS-control with TBS to a proximal face-selective cortical region . We predicted that TBS to OPA (but not OFA) would lead to decreased responses to scenes and buildings (but not other categories) in other scene-selective cortical regions. Across both ROI and whole-volume analyses, we observed decreased responses to scenes in PPA as a result of TBS. However, these effects were neither category specific, with decreased responses to all stimulus categories, nor limited to scene-selective regions, with decreases also observed in face-selective fusiform face area (FFA). Furthermore, similar effects were observed with TBS to OFA, thus effects were not specific to the stimulation site in the lateral occipital cortex. Whilst these data are suggestive of a causal, but non-specific relationship between lateral occipital and ventral temporal cortex, we discuss several factors that could have underpinned this result, such as the differences between TBS and online TMS, the role of anatomical distance between stimulated regions and how TMS effects are operationalised. Furthermore, our findings highlight the importance of active control conditions in brain stimulation experiments to accurately assess functional and causal connectivity between specific brain regions. These eIn particular, recent work has explored this question with respect to the face-selective network using consecutive TBS/fMRI . Here, iHere, we sought to investigate how causal intervention affects visual information processing across scene-selective regions. We compared TBS of OPA with both a no-stimulation control condition (Sham) and an active control condition (nearby face selective OFA) and measured BOLD responses to a range of stimulus categories across lateral occipital and ventral temporal cortex. We hypothesized that TBS of OPA, but not TBS of OFA or Sham, would lead to reduced response to scenes, but not other stimulus categories, in OPA as well as downstream scene-selective regions PPA and MPA.2.2.1.n = 16, 12 females, mean age = 24.4 years). All participants had normal or corrected to normal vision and gave written informed consent. We tested 16 participants based on a survey of related studies. As far as we are aware no prior study had stimulated OPA with theta-burst stimulation making it hard to conduct a formal power analysis. Prior studies stimulating OPA and reporting effects measured with fMRI used a 1 Hz TMS protocol with sample sizes of 8 (n = 15). The National Institutes of Health Institutional Review Board approved the consent and protocol. This work was supported by the Intramural Research program of the National Institutes of Health – National Institute of Mental Health Clinical Study Protocols 93-M-0170 and 12-M-0128, NCT00001360.A total of 16 participants completed the experiments  mapping session, followed by three counterbalanced TBS/fMRI sessions: TBS to OPA, TBS to OFA (active control condition) and Sham (no-stimulation control condition). The localizer/pRF mapping session was conducted first. Next, the order of the TBS/fMRI sessions was counterbalanced in a nested way, such that half of the participants received TBS to OPA before TBS to OFA, and half of the participants received Sham before active TBS  and all TBS/fMRI scans (n = 16) were conducted on a 3.0T GE Sigma MRI scanner in the Clinical Research Center on the National Institutes of Health campus . Whole-brain volumes were acquired using an eight-channel head coil . In each TBS/fMRI scanning session, two T1-weighted anatomical images were acquired using the magnetization-prepared rapid gradient echo (MPRAGE) sequence .Initial functional localiser scans (2.2.2.n = 2) were conducted on a 7.0T Siemens scanner (gradient echo planar imaging (EPI) sequence with a 32-channel head coil .Initial functional localiser scans . During category-localizer runs, color images from six categories  were presented at fixation in 16 s blocks , whilst participants performed a one-back task. Blocks were separated by 4 s blanks and started and ended with a 16 s baseline period. The total run length was 279 s (~4.7 min). Each category was presented twice per run, with the order of presentation counterbalanced across participants and runs. Participants performed a one-back task. For the remaining two participants, initial localization scans (4 runs) were completed on a 7.0T scanner. During category-localizer runs, grayscale images from eight categories  were presented at fixation in 16 s blocks , whilst participants performed a one-back task .2.2.4.During pRF mapping, participants fixated centrally whilst a bar aperture traversed gradually throughout the visual field revealing scene fragments. During each run the bar aperture made eight sweeps through the visual field  and participants indicated a change in fixation color via a button press see .2.2.5.Across the three TBS/fMRI sessions  participants fixated a central cross whilst grayscale images  from eight different categories  were back projected on a screen mounted onto the head coil with 1024 × 768 pixel resolution . Images 2.2.6.All three TMS/fMRI scanning sessions followed an identical design . After i2.2.7.A Magstim Super Rapid 2 stimulator  was used to deliver TBS using a figure-of-eight coil (70 mm wing diameter). TBS was delivered at 30% of maximum machine output over the target voxel for each session. A continuous TBS paradigm  was used2.3.SCR_005927). Below we outline the pre-processing steps taken for both the initial functional localization and for the TBS/fMRI experiments.All anatomical and functional data were pre-processed and analyzed using AFNI  (RRID: S2.3.1.3dVolreg) after removal of the appropriate dummy volumes to allow stabilization of the magnetic field. Following motion correction, images were spatially smoothed (3dmerge) with a 5 mm full-width-half-maximum smoothing kernel. Signal amplitudes were then converted into percent signal change (3dTstat). We employed a general linear model implemented in AFNI . The data at each time point were treated as the sum of all effects thought to be present at that time and the time-series was compared against a Generalized Least Squares (GSLQ) model fit with REML estimation of the temporal auto-correlation structure. Responses were modelled by convolving a standard gamma function with a 16 s square wave for each stimulus block. Estimated motion parameters were included as additional regressors of no-interest and fourth-order polynomials were included to account for slow drifts in the MR signal over time. To derive response magnitude per category, t-tests were performed between the category-specific beta coefficients and baseline.All images were motion corrected to the first volume of the first run (using the AFNI function 2.3.2.3dVolreg) and detrended (3dDetrend) before being averaged across runs. The average time-series data were analysed using a 2-dimensional Gaussian pRF model implemented in AFNI. A detailed description of the model is available elsewhere . This anatomical image is assumed to be half-way between the anatomical images acquired at the start and end of each session and therefore constitutes a reference image that is unbiased to either the Pre or the Post session. Once pre-processed, all EPI data within a session were then deobliqued (3dWarp) and aligned to this mean anatomical image . GLMs were estimated for each run separately  as opposed to concatenating all runs together before statistical analysis (default option in AFNI) in the unaligned, native volume space, after which the resulting statistical parametric maps were aligned to the mean anatomical image by applying the transformation matrices from the EPI alignment.Functional data from TMS sessions followed a similar pre-processing pipeline to that specified above, but differed in the following ways. For each TBS/fMRI session a mean anatomical image was first computed across the two T1 scans acquired before (Pre) and after (Post) TBS . Stimulation sites for subsequent TBS sessions were identified on an individual participant basis using the Brainsight TMS-MRI co-registration system. In each participant, the statistical maps reflecting the contrast of Scenes versus Faces were overlaid onto the high-resolution anatomical scan. The primary OPA stimulation site was defined as the peak voxel of scene-selectivity within right OPA, and the active-control OFA stimulation site as the peak voxel of face-selectivity within right OFA. Individual target sites for each participant are depicted in Scene- and face-selective ROIs . Neither the effect of TMS condition =1.98, p = 0.15) nor any pairwise comparisons were significant (p>0.05). The lack of a TMS condition effect indicates an equivalent level of precision across the three conditions. This is valuable data as the Sham condition targeted the OPA and thus no difference between these conditions  would be expected. Coil-target distance measures the Euclidean distance between the target voxel and the calibration point of the TMS coil. For each TMS condition  we calculated the mean coil-target distance in each participant and submitted these to a linear-mixed model with TMS condition as the only main effect. Neither the main effect of TMS condition =1.02, p = 0.37) nor any pairwise-comparisons (p>0.05) were significant. Although it is possible that a given target is further away (i.e. deeper in a sulcus) than another, the lack of a significant TMS condition effect suggests a) that our target sites were similarly distanced from the coil and b) that our calibration procedure was consistent across TBS/fMRI sessions.We took advantage of two measurements  of each individual participant’s hemispheres derived from the Freesurfer4 autorecon script (http://surfer.nmr.mgh.harvard.edu/) using the Surface Mapping with AFNI (SUMA) software. The freesurfer reconstructions were based on the T1 scan obtained in the localizer session. In order to align the functional data to these surfaces, the mean (Pre-Post) T1 from each TBS/fMRI session was first aligned to the volume used for surface reconstruction (@SUMA_AlignToExperiment).In each participant, the pre-processed functional data from all sessions were projected onto surface reconstructions . ROIs were defined using the interactive ROI drawing tool in SUMA according to both these statistical criteria and accepted anatomical locations . No further anatomical or functional constraints were applied.ROIs were defined by overlaying the statistical results of the contrast Scenes versus Faces  onto the surface reconstructions of each individual participant, before thresholding (2.5.3.ROI2dataset), which was subsequently used to extract beta-coefficients for each stimulus category from the three separate TBS/fMRI sessions for each surface node within the ROI (ConvertDset). We verified that the localizer ROIs were adequately aligned with the experimental runs by visual inspection and by computing the Euclidean distance between the centers of mass of the localizer ROI and the Pre-TBS runs in each individual participant (see lme4 package implemented in R (https://osf.io/6nq7r/). Our primary analyses modelled participants as a random effect using a random intercept term. Additional analyses included random effects for the within-subject factors (i.e. random slopes), and formal model comparison using Likelihood Ratio Tests .To investigate whole brain effects of TMS we employed a linear mixed effects model in AFNI 3dLME;  in each 3.3.1.To assess the causal and functional relationships between scene-selective regions, fMRI BOLD responses to scene, building, face and object stimuli were measured in OPA, PPA and MPA before and after application of TBS to OPA. To control for non-TBS induced effects, the same participants also took part in an additional session in which the exact same procedure was followed but no TBS was applied  stimulus-specific, occurring for preferred stimuli only; and 2) stimulation site-specific, occurring only as a result of stimulating the region selective to the preferred stimulus, but not a region selective to another category.. These two types of category-specificity could be revealed by our experimental design in the following ways. First, if TMS effects in scene-selective cortex are both stimulus- and site-specific, we should observe a decreased response to scene and building stimuli (but not face or object stimuli) as a result of TBS to OPA (but not OFA). Second, if TMS effects are site-specific but not stimulus-specific, stimulation to OPA (but not OFA) will cause scene-selective regions to respond less to We assessed the presence and category-specificity of TBS-induced response reductions in each scene-selective ROI using a linear mixed effects model see  that tes3.2.p = 0.86), nor any two-way interactions , reflecting the well-known category-selectivity of OPA; as shown in In OPA, contrary to our predictions, we found no evidence for an interaction between TMS-condition, Session and Stimulus  = 0.61, p = 1; p < 0.001, Bonferroni-corrected). As shown in In PPA, similarly no evidence was found for a three-way interaction between TMS-condition, Session and Stimulus  = 0.26, In MPA, no significant interactions related to TMS stimulation were observed, even though this region exhibited robust visual responses to scene stimuli in accordance with its category preference see .p < 0.001, Bonferroni-corrected) in the downstream region  and the FFA. Interestingly, these ROIs showed a similar pattern as for the scene-selective regions: a lack of a local TBS effect at the stimulation ROI OFA; -B coupleion FFA; -D indicaTo examine potential effects of TBS outside category-selective cortex, we also examined responses in retinotopically defined V1 corresponding to the stimulus size. As expected, we observed robust visual responses to all stimulus categories, but no interaction effects related to TMS see .p < 0.0001) was found in all ROIs; further data inspection indicated that this main effect reflected an overall decrease in response over time within both Pre and Post sessions.Finally, while none of the analyses indicated any interactions with Run indicative of a temporally specific TBS effect, a strong main effect of Run  > 41.1, all Taken together, the ROI analyses demonstrate effects of TMS to lateral occipital cortex in both scene- and face-selective downstream ventral temporal regions. However, these effects were neither stimulus- nor site-specific. While the reduced responses appear robust in ventral regions, especially for OFA stimulation, one puzzling aspect of the results is the lack of reduced activity in the stimulated lateral occipital ROIs themselves. One possibility is that we overlooked a local effect within the stimulated ROIs themselves. To further clarify this pattern, we next proceeded to fit a whole-brain linear mixed effects model that is not constrained to average effects across cortical regions.3.3.a priori defined ROIs. These analyses were conducted on the surface data (in each hemisphere separately) of the stimulated hemisphere  and thus averaging could be masking local effects of TMS within the ROIs. To avoid the issue, we performed a linear mixed effects analysis at the whole brain level (implemented in AFNI using 3dLME), which has the potential to reveal local effects of TMS if spatially consistent across participants as well as potential downstream effects that fall outside our q = 0.000042). The Session by Run interaction revealed only a very small cluster on the ventral surface, close to the posterior boundary of PPA, which reflects on average a larger reduction in response at this location across runs in the Post-TBS sessions.At the whole brain level, significant clusters were evident for each main effect see , but onlIn line with our ROI analyses, we focused on the Session by TMS-condition interaction as it demonstrates differences in TBS-induced effects across sessions . Significant interaction effects were observed as localised clusters that either overlapped with, or were in close proximity to, our group-based definitions for OPA, OFA, PPA, FFA and parts of V1 . While nTo understand what was driving the interaction in these clusters, we looked at the direction of the difference in activation across Sessions (Post - Pre), for each TMS condition separately. The group averaged maps are presented in During Sham baseline, effects in each ROI were almost exclusively positive , reflecting on average an increased response during the Post session. In downstream ventral temporal regions PPA and FFA, consistent with the ROI analyses, the interaction appears to be driven by reduced responses as a result of either stimulating OPA or OFA. In the stimulated regions, the pattern is slightly different for each ROI. For clusters within both OPA and OFA there were decreased responses in a local region for OPA stimulation but increased responses for OFA stimulation. However, in OPA but not OFA these increases were greater than those for Sham.Overall, the whole-brain analyses show that whilst TMS of both OPA and OFA elicited an overall reduction in response relative to the Sham baseline, the impact of stimulation also differed between stimulation sites. Relative to Sham, OPA stimulation led to a local decrease within OPA, OFA, PPA and FFA, whereas OFA stimulation led to a local increase in response in OPA, a small decrease in OFA, and a more pronounced reduction in downstream areas PPA and FFA.3.4.This study was not optimized to examine the behavioral impact of brain stimulation. However, given the widespread nature of the observed TBS effects, we performed an exploratory analysis to test whether participant performance was affected by TBS, considering both 2-back repeat performance and reaction times see . No beha4.Our results show that theta-burst stimulation of a scene-selective region in the lateral occipital cortex (OPA) reduces BOLD responses in a scene-selective region in the ventral temporal cortex (PPA). Although we did not observe effects of TBS in OPA itself when conducting a ROI-based analysis that averaged activation values across the entire ROI, the whole-brain analysis did reveal the presence of a local effect within OPA as a result of stimulation. While we thus observed both local and downstream effects of TMS on fMRI responses in scene regions to visual stimuli, our findings did not fully confirm our predictions. Specifically, we predicted that TMS would reduce responses in downstream scene-selective regions in a stimulus- and stimulation-site specific manner. However, responses in PPA were reduced across all stimulus categories, and were similarly reduced when stimulating an active control region that was face-selective (OFA). Conversely, TBS-induced effects were observed in face-selective FFA when stimulating not only face-selective OFA but also when stimulating scene-selective OPA. Furthermore, only limited TMS-induced effects were observed in the proximity of a second downstream scene-selective region, MPA. Collectively, these results show that effects of theta-burst TMS to the lateral occipital cortex are not constrained to a single category-selective network of regions. Below we discuss the implications of these results further and highlight the importance of an active control site in studies such as this.4.1.Our results clearly indicate that stimulation of both OPA and OFA causally decreased responses relative to the Sham baseline. The OFA stimulation served primarily as a control condition for our main research question investigating causal interactions between scene-selective regions OPA, PPA and MPA, and our experiment was designed to investigate scene processing. However, we also investigated TBS induced effects in FFA, which showed that stimulating OPA in fact also reduced responses in FFA (similar to stimulating OFA). The fact that TMS of both OPA and OFA elicited downstream reductions in the same areas speaks to whether or not these scene- and face-selective networks can be separated cleanly with theta-burst stimulation and fMRI. Online TMS experiments  have demSome evidence for separation between regions using TBS/fMRI has been shown within the face-network ; 2017 usA further possibility for the lack of site-specific effects in the current study is how the impact of TBS was operationalised. Here, our principal method for assessing the impact of TBS was a comparison of the fMRI responses before and after stimulation and not its impact on behavioural performance, which is often how TMS effects are demonstrated . We knowInterestingly, TMS induced reductions in ventral temporal cortex appeared slightly stronger overall for stimulating OFA compared to stimulating OPA. It is possible that the strong impact of OFA stimulation on downstream areas of the ventral temporal cortex reflects its close proximity to VTC . In all participants, OFA is located ventral and slightly anterior to OPA. Indeed, in many cases there is little cortex separating OFA on the lateral surface and FFA on the ventral surface. Any impact of OPA stimulation has much further to travel to affect VTC regions. Consistent with this suggestion, prior work  reportedWe also assessed whether the lack of site-specific effects was due to the two target sites being in closer proximity to each other in certain participants over others. To look at this, we calculated the Euclidean distance between target sites in each participant . For 15/16 participants, these distances were largely similar. Indeed, only a single participant had a distance > 1 std above the mean. Thus our results are unlikely to reflect closer proximity of target locations in a subset of participants.4.2.Both the ROI-based and whole-brain analysis did not indicate statistical evidence for stimulus-specificity of the TBS-induced effects, suggesting that TBS similarly affected fMRI responses to all stimulus categories, rather than the specific category preferred by either the stimulated ROI or the downstream ROI. Prior evidence for stimulus-specific effects of TMS on fMRI responses is mixed. Stimulation of face-selective OFA has been found to reduce responses in downstream ventral temporal regions for both static faces and objects , but ano4.3.Whilst TBS-induced effects were observed in downstream PPA, they were largely absent in MPA . There are two, not necessarily mutually exclusive, explanations for the lack of TBS-induced effect in MPA. First, it is possible that this lack of effect reflects the general weaker responses in MPA relative to the other ROIs . Indeed,4.4.Two types of controls were employed in order to test the specificity of TMS stimulation (compared to Sham), and the specificity of stimulating the scene-network . The inclusion of a Sham condition allowed us to test the effect of stimulation, but the active control site (OFA) allowed us to test whether any stimulation effects were specific to the scene-network. It is important to note that our interpretations would be dramatically different without the inclusion of this active control site. If we had only compared OPA stimulation with Sham, we would have reported that TBS to OPA leads to both a local decrease within OPA itself, as well as downstream decreases in PPA, and we could have interpreted this as evidence for a key role of OPA in routing visual information through the scene network. Such an interpretation however is not valid when the results from our active control are taken into account. Here, we saw that reductions in PPA were also evident when stimulating face-selective OFA, suggesting that causal effects in scene-selective ventral temporal cortex can also be elicited by stimulating visually responsive regions that do not share the same category preference. The same pattern was observed for reductions in face-selective ventral temporal cortex (FFA) when stimulating scene-selective lateral-occipital cortex.Whilst this makes the interpretation more challenging, it is important to reflect on the potentially misleading picture that would have been painted had we not included an active control condition. Recent TMS work, both behaviourally  and in c4.5.one-tailed t-test: t(15) = 1.96, p<0.03; Our whole brain analysis revealed a highly localised TBS effect within OPA. In OFA, however, the interaction effect was weaker in magnitude and less localised to our group-based definition. We considered the possibility that this might simply reflect less variability in stimulation site for OPA across participants as compared to OFA. To address this, we assessed the pairwise Euclidean distances between stimulation sites across participants and found that there was significantly less variance in stimulation location across participants for OPA compared to OFA =0.91, p = 0.37). Third, we calculated the mean Euclidean distance between the target voxel in each ROI and the peak voxel from the equivalent contrast in each of the four Pre Sham runs, before averaging these values across runs. On average, displacements were ~10 mm for both target sites (t-test showed no significant difference in shift distance (t(15)=0.08, p = 0.93). Taken together, we are confident that the lack of local effect in OFA is not due to inaccuracies in localization or alignment across sessions.Alternatively, it is possible that inaccuracies in ROI alignment between sessions could lead to responses being shifted away from our original OFA definitions. To address this possibility we conducted several complementary analyses, which we believe rule out this possibility and provide confidence in our analysis approach. First, we examined the spatial overlap between our original ROI definitions  and those computed from each of the four Pre TBS runs. Despite being computed on individual runs as opposed to the average of six, all Pre TBS ROIs fell within the boundaries of our original ROI definitions, although considerably smaller in areal extent . Second,et sites . Indeed,4.6.The choice of TMS protocol can impact the spatial extent , tempora4.7.Although it is common practice in fMRI research to have participants perform the same tasks in multiple runs, it is not without its drawbacks. Prior work  demonstrLocal effects of TBS stimulation to scene- and face-selective cortex were observed within the stimulated regions, but these were insensitive to the preferred category of the stimulated region. Downstream effects were observed to be focal to scene and face-selective regions, but these effects were not constrained by the preferred category of the stimulated region, since similar TMS effects were observed when stimulating either a scene- or a face-selective lateral occipital region. Collectively, these results show that effects of theta-burst TMS to the lateral occipital cortex are not constrained to a single category-selective network, suggesting that information processing in different groups of category-selective regions  may be less independent that one might assume based on their distinct category preferences alone. It is vital however to also recognize that the evidence for the independence of these different category-selective networks changes with experimental decisions, such as the type of TMS and how such TMS effects are operationalised. Finally, our results highlight the importance of a close proximity control stimulation site to fully interpret the site-specificity of TMS effects.1


This article quantifies changes in employment and average wages of employees of 6 key health care organizations during the COVID-19 pandemic. Understanding trends in employment levels by health care settings and locations is critical for planning and responding to public health crises.The COVID-19 pandemic has disrupted the US health care workforce owing to changes in the use and finances of health care clinician offices and institutions, increased health risks, burnout from increased patient burdens, and child care disruptions.5 We conducted 2 analyses to quantify changes in employment and average wages of employees of 6 key health care organizations  during 2020 and the first 6 months of 2021. First, we examined quarterly national trends in health care employment and average wages between March 31, 2020 (2020-Q1), and June 30, 2021 (2021-Q2), relative to 2019 (pre-COVID levels). Second, we examined associations between the 12-month changes in employment levels during 2019 to 2021, COVID-19 burden, and pre-COVID physician-to-population ratio. Multivariable linear regression models were conducted with Stata, version 17.0. The study was granted Not Regulated status by the University of Michigan Medical School Institutional Review Board (HUM00207016).We used industry- and county-level data from the US Bureau of Labor Statistics Quarterly Census of Employment and Wages .Health care employment levels declined suddenly, from 22.2 million in 2019 to 21.1 million, in 2020-Q2—a 5.2% decline , and considerably rebounded to 21.8 million in 2021-Q2. Average wages within the health care sector increased at a lower rate relative to all industries’ changes .Employment declines varied by types of health care organizations , with thP = .008). Compared with the top 20%, counties with the lowest physician-to-population ratios tended to have higher adjusted employment levels in offices of physicians  and among offices of dentists . These associations were not statistically significant among other types of institutions.The 6 We documented changes in health care employment levels and wages during 2020 and 2021. We found substantial employment declines among SNFs, which were more severe in counties with high COVID-19 burden. The 2020 declines in employment among offices of physicians and offices of dentists were relatively smaller in counties with lower pre-pandemic physician-to-population ratios.Protecting the health care sector has been a priority in the pandemic.Limitations include the exclusion of certain counties with censored data and our inability to examine mechanisms for workforce changes. Future research is needed to understand if organizations are demanding fewer workers or fewer workers are willing to work at health care positions. Overall, our results imply that intensified early efforts are needed to protect the health care workforce in future pandemics.