Hypertensive heart disease (HHD) develops in response to the chronic exposure of the left ventricle and left atrium to elevated systemic blood pressure. Left ventricular structural changes include hypertrophy and interstitial fibrosis that in turn lead to functional changes including diastolic dysfunction and impaired left atrial and LV mechanical function. Ultimately, these changes can lead to heart failure with a preserved (HFpEF) or reduced (HFrEF) ejection fraction. This review will outline the clinical evaluation of a patient with hypertension and/or suspected HHD, with a particular emphasis on the role and recent advances of multimodality imaging in both diagnosis and differential diagnosis. Worldwide, the age-standardised prevalence of hypertension continues to rise and has reached 30% in the general population and up to two-thirds in those over the age of 60 ,2. HyperLeft ventricular hypertrophy (LVH)—defined as an increase in the LV wall thickness or mass—is widely regarded as a cardinal feature and phase in the evolution of HHD . TraditiLV structural changes include hypertrophy and interstitial fibrosis that in turn lead to functional changes including diastolic dysfunction and impaired LA and LV mechanical function. Ultimately, these changes can lead to heart failure with a preserved (HFpEF) or reduced (HFrEF) ejection fraction . It shouMost patients with hypertension are asymptomatic and, as such, the condition has often been described as a silent killer . HistoryAll patients should have a baseline 12-lead Electrocardiogram to screen for LVH/end-organ damage although it has a low sensitivity for detecting LVH . All patGiven its availability and low cost, transthoracic echocardiography (TTE) is the first-line diagnostic imaging test to detect structural and functional changes in HHD. Left ventricular mass index (LVMI), left ventricular diastolic dysfunction (LVDD), and impaired global longitudinal strain (GLS) have prognostic implications in patients with arterial hypertension ,10,11. HEchocardiographic assessment of LVH and thus left ventricular mass (LVM) relies on linear measurements of LV dimensions on 2-dimensional images. Calculation of myocardial mass then uses geometric assumptions with respect to LV shape , which, LV systolic function is maintained at normal or near-normal values until the late stages of HHD. The most commonly used surrogate marker of LV systolic function is left ventricular ejection fraction (LVEF), which depends not only on LV contractility but also on pre- and afterload and left atrial function. In clinical practice, LVEF is measured on 2D echocardiography using Simpson’s biplane method or on 3D echocardiography using manually assisted semi-automated endocardial border tracking . Of noteWhile LVEF may remain normal along the natural history of HHD, echocardiographic techniques such as speckle tracking strain have been shown to be able to detect subtler impairments in myocardial mechanics such as reduced global longitudinal strain (GLS) . In hypeInherent to the LV structural changes including hypertrophy and interstitial fibrosis, LV elasticity, compliance, and stiffness are compromised in hypertensive patients and lead to LVDD. While a complete description of an echocardiographic assessment of diastolic function is beyond the scope of the present review, measurements include Doppler and tissue Doppler measurements of mitral inflow and mitral annular velocities . These mThe LA dilates as a consequence of LVDD in HHD, and the LA volume index (LAVI) has been shown to independently predict cardiovascular events . RecentlFurthermore, arterial hypertension is a risk factor for developing an abdominal aortic aneurysm. Screening for abdominal aortic aneurysms can easily be integrated into a routine echocardiography assessment and is indicated in patients with long-standing hypertension and individuals over 65 years of age  .Serial echocardiography can be used in principle to monitor longitudinal changes in LV mass in response to treatment, which may have prognostic implications; however, the higher accuracy and precision of CMR make this more suited for this application.Hypertensive patients with LV hypertrophy disproportionate to the degree of hypertension may be referred for CMR for further examination and differential diagnosis. CMR is regarded as the non-invasive gold standard for the evaluation of LV volumes, systolic function, LV mass, and myocardial tissue characterisation . Image aSimilar to echocardiography, LV geometry can be assessed using relative wall thickness (RWT) and CMR-specific normal values for LV myocardial mass . LA dimeFeature-tracking strain imaging can also be performed in CMR with the advantage of better image quality, lack of angle dependency or impediment from difficult acoustic windows, and less observer dependency . MyocardHypertension is a well-established atherosclerotic risk factor for the evolution of coronary artery disease (CAD). Both macrovascular CAD of the epicardial coronary arteries and microvascular CAD  are detectable by CMR stress perfusion . There iCMR stress perfusion essentially visualizes myocardial first-pass perfusion during pharmacological stress. Images are interpreted in conjunction with rest perfusion images and late gadolinium enhancement (LGE) images. In the presence of a significant epicardial coronary stenosis, the myocardial contrast uptake is reduced in a specific coronary territory (coronary pattern). Conversely, microvascular dysfunction displays a more diffuse and delayed but synchronous myocardial contrast uptake during stress. Stress CMR offers excellent sensitivity and specificity for the detection of anatomically and functionally significant CAD  and alloTo avoid the low but important risks of endomyocardial biopsy, which can have an overall complication rate of up to 6% , myocardLGE has become the reference standard for non-invasive imaging of myocardial scar and focal fibrosis . GadolinT1 before and after the administration of contrast agents, as well as the patient’s haematocrit. ECV can then be calculated using the formula:LGE allows the detection of focal alterations in the myocardium, but diffuse fibrosis may go undetected on LGE imaging. Tissue characterisation with parametric mapping methods such as T1 and ECV mapping has the potential to detect and quantify both focal and diffuse alterations in the myocardial structure. Furthermore, changes in the myocardium over time may be assessed longitudinally . EstimatECV values vary between 25.3 and 3.5% . C,6. CECV  methods . FurtherT1 and ECV has been shown to have incremental diagnostic benefits even in very early disease stages , there is an overlap between different cardiomyopathies and some overlap with normal T1 values. The difference in ECV between normal subjects and patients with HHD is small (0.29 ± 0.03 vs. 0.27 ± 0.02) , a, a68], aSarcoid can occasionally cause LV thickening when there is focal or extensive granulomatous infiltration of the myocardium . If therThe extent/severity of hypertrophy can be of value in a differential diagnosis. It is unusual for hypertensive heart disease to produce very severe hypertrophy (>20 mm) and never extreme hypertrophy (>30 mm). Similarly, it is often suggested that high levels of athletic activity can be associated with LVH. However, most patients who experience athletic remodelling develop mild symmetrical cavity dilatation and modest increases in LV mass. Increases in LV wall thickness are uncommon and, if present, tend to be very mild (13–15 mm). Such changes are more commonly seen in athletes with black African ancestry. Increases in LV wall thickness can often be seen in recreational bodybuilders who take androgens or an exogenous growth hormone . InteresAs LVH increases, there is a reduction in ECV in athletes (cellular hypertrophy) but an increase in ECV in patients with HCM (cellular disarray and extracellular matrix expansion). Based on this divergent finding, ECV may be helpful to distinguish HCM and athletic remodelling, particularly in subjects with indeterminate maximal wall thickness (13–15 mm) .p < 0.05) [The relationship between hypertension grade and coronary atherosclerosis was evaluated in 8238 patients who underwent calcium scoring and computed tomography coronary angiography (CTCA) for screening purposes. The prevalence of calcified and non-calcified plaques increased with the grade of hypertension and was 17%, 28%, 34%, and 40% in normotensive, pre-hypertension, stage 1 hypertension, and stage 2 hypertension, respectively ( < 0.05) .CTCA has the highest negative predictive value of all imaging techniques regarding the exclusion of macrovascular CAD. Often coronary artery tortuosity can be documented M. ArteriThe correlation between blood pressure and myocardial metabolism has been evaluated in 86 individuals undergoing fluorodeoxyglucose positron emission tomography (18-F-FDG-PET) PET . This isApart from diagnosing macrovascular disease/ischaemia in patients with HHD, myocardial perfusion reserve (MPR) assessed by Rubidium (Rb)-82 PET can be used to risk stratify patients with hypertension. In a study of 517 hypertensive patients undergoing Rb-PET imaging, the 26% of patients who had resistant hypertension had more frequent LVH, lower myocardial blood flow rates, and lower MPR than subjects without resistant hypertension. Age, resistant hypertension, and impaired MPR were independent predictors of adverse events over a median follow-up of 38 months .Secondary hypertension affects approximately 5–10% of the general hypertensive population . ImagingThe most common cause of primary hyperaldosteronism is an aldosterone-producing adrenal adenoma. Adrenal adenomas usually present as round or oval masses. Using a threshold value of ≤10 Hounsfield units (HU) at non-contrast CT leads to high sensitivity and specificity in their detection . Given tPhaeochromocytomas are a rare cause of secondary hypertension and account for only <5% of patients with secondary hypertension. These tumours usually present as large, heterogeneous masses with both necrosis and cystic changes and avid contrast enhancement .Common non-endocrine causes are aortic coarctation N and renAortic coarctation is defined as luminal narrowing near the origin of the left subclavian artery and ligamentum arteriosum and is a very rare cause of secondary hypertension with only 0.2% of patients with hypertension affected . IncreasRegarding renovascular hypertension, FMD or renal artery stenosis are the most common causes O. Both cPatients with long-standing hypertension often also develop increased arterial stiffness with accelerated aortic pulse wave velocity as measured by CMR velocity-encoded imaging. A stiff aorta may further increase LV afterload .All markers of cardiac damage in HHD are influenced by age, gender, and ethnicity. Normalisation to body surface area may be misleading in obese individuals; normalisation for body height, a good surrogate of fat-free mass, appears to be more acceptable but used less frequently in clinical practice . All imaDue to the high prevalence of hypertension, HHD is one of the most common heart diseases and a frequent cause of diastolic heart failure and LV impairment. In clinical practice, comprehensive echocardiography may be sufficient to recognize most features of HHD. In patients with LV hypertrophy disproportionate to the degree of hypertension and patients where dual pathology is assumed, tissue characterization with CMR can be very helpful in the differential diagnosis, risk stratification, and also to detect extracardiac pathology associated with HHD.


Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a systemic disorder in which multi-organ dysfunction may occur from mitochondrial metabolism failure. Maternally inherited mutations in the MT-TL1 gene are the most frequent causes for this disorder. Clinical manifestations may include stroke-like episodes, epilepsy, dementia, headache and myopathy. Among these, acute visual failure, usually in association with cortical blindness, can occur because of stroke-like episodes affecting the occipital cortex or the visual pathways. Vision loss due to optic neuropathy is otherwise considered a typical manifestation of other mitochondrial diseases such as Leber hereditary optic neuropathy (LHON).Here we describe a 55-year-old woman, sister of a previously described patient with MELAS harbouring the m.3243A > G  mutation, with otherwise unremarkable medical history, that presented with subacute, painful visual impairment of one eye, accompanied by proximal muscular pain and headache. Over the next weeks, she developed severe and progressive vision loss limited to one eye. Ocular examination confirmed unilateral swelling of the optic nerve head; fluorescein angiography showed segmental perfusion delay in the optic disc and papillary leakage. Neuroimaging, blood and CSF examination and temporal artery biopsy ruled out neuroinflammatory disorders and giant cell arteritis (GCA). Mitochondrial sequencing analysis confirmed the m.3243A > G transition, and excluded the three most common LHON mutations, as well as the m.3376G > A LHON/MELAS overlap syndrome mutation. Based on the constellation of clinical symptoms and signs presented in our patient, including the muscular involvement, and the results of the investigations, the diagnosis of optic neuropathy as a stroke-like event affecting the optic disc was performed. L-arginine and ubidecarenone therapies were started with the aim to improve stroke-like episode symptoms and prevention. The visual defect remained stable with no further progression or outbreak of new symptoms.Atypical clinical presentations must be always considered in mitochondrial disorders, even in well-described phenotypes and when mutational load in peripheral tissue is low. Mitotic segregation of mitochondrial DNA (mtDNA) does not allow to know the exact degree of heteroplasmy existent within different tissue, such as retina and optic nerve. Important therapeutic implications arise from a correct diagnosis of atypical presentation of mitochondrial disorders.The online version contains supplementary material available at 10.1186/s12883-023-03198-3. Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) is one of the most common maternally inherited mitochondrial diseases –4.Although childhood represents the typical age of onset, in a small percentage of cases the disease starts adulthood and is commonly associated with better prognosis and a relatively less aggressive clinical course . The preLeu(UUR). Besides that, other different pathogenic mutations have been described [An A to G transition in the 3243 position of mitochondrial DNA (mtDNA) is responsible for up to 80% of cases . Positioescribed , 2, 6. Aescribed . Lactic Different plausible theories have been proposed to explain the pathogenic mechanism underlying stroke-like episodes (SLEs) in MELAS. According to a metabolic hypothesis, a local breakdown of the mitochondrial energy metabolism occurs, either in neurons, glial cells, or in cells constituting the blood brain barrier (BBB) , 3. A vaIncreased signal on diffusion-weighted imaging represents a characteristic finding in the magnetic resonance imaging (MRI) of the acute stroke-like events, accompanied by high signal on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences . InteresVisual impairment represents a possible manifestation of MELAS. Retinal dystrophy, pigmentary retinopathy, ptosis, ophthalmoplegia, optic atrophy, and corneal polymegathism are well described ophthalmologic expressions of the syndrome that can cause visual defects , 8. AcutA 55-year-old woman with known medical history of arterial hypertension, hypercholesterolemia and aortic stenosis, presented with a subacute episode of visual loss started as an arcuate visual field defect in the left eye (LE) and evolved in the next days in a larger central defect. Visual symptoms were accompanied by the onset headache and diffuse muscular pain, in particular with proximal musculature involvement. Her younger brother was diagnosed a few years earlier with MELAS for relapsing episodes of intestinal pseudo-obstruction, followed by SLEs. In a short period of time he experienced a rapidly progressive cognitive decline, unfortunately leading to his death . He carrAt the follow up visit after four months, visual field defects remained stable Fig. B, while Here we present a unique phenotype of MELAS related optic neuropathy in a patient with a common m.3243A > G mutation. The visual defect and the ophthalmologic alteration compatible with ischemic neuropathy may represent the consequence of energy deficiency in the optic nerve due to a MELAS vascular related SLE. mtDNA sequencing was important to confirm MELAS diagnosis and to rule out possible mutations which account for MELAS/LHON overlap syndrome. However, some aspects regarding the onset of the visual defect were atypical for a LHON: severe unilateral optic nerve head swelling, segmental perfusion delay and leakage of the optic disc on FA were more consistent with an ischemic optic neuropathy, in addition orbital pain exacerbated by ocular movements combined with throbbing temporal pain worsened by chewing were suggestive of an arteritic aetiology. The diffuse muscular pain that she reported was a typical manifestation that could either be associated to the myopathy existing within MELAS, either to inflammatory phenomena affecting muscles during GCA. PET and temporal artery biopsy were indeed fundamental to exclude a large vessel disease vasculitis. ESR and CRP were unreliable since they were first assessed after ten days of steroids therapy. Excluding such type of vasculitis, it was possible to start prednisone titration in a patient harbouring a syndrome known to be associated with possible endocrinological dysfunction (such as diabetes) which could also be aggravated by steroids.Although visual evoked potential showing a reduction in amplitude with prolonged P100 in the left eye represents a typical finding of demyelinating lesion affecting the optic nerve, brain MRI did not show any altered signal of the optic nerve, which would otherwise be present in the context of MS, NMO and NMOSD presentation.. In our patient, who presented risk factors for NAION, the perfusion of the optic disc could have been partially compromised by hypertension and worsened because of SLE secondary to a microangiopathy or transient local energetic breakdown in mitochondrial function. MELAS-like retinal anomalies detected on fundoscopy and fundus autofluorescence (FAF) imaging, further support this theory [SLEs are commonly accompanied by elevated lactate levels in serum and CSF analysis, along with increased lactate at MR-spectroscopy in the central nervous system (CNS) regions affected by the SLE event , 3, 7. Is theory , 14. Mors theory . Based oThis is the second case of a possible SLE described in a carrier of mutation m.3243A > G with swollen optic disc; previously, a case with bilateral transient optic disc oedema was described . HoweverIn conclusion, atypical clinical presentations must be always considered in mitochondrial disorders, even in well-described phenotypes and even if mutational load in peripheral tissue  is low. Indeed, the stochastic mitotic segregation of mtDNA molecules does not allow to foresee the exact degree of heteroplasmy existent within different tissues, such as retina and optic nerve. As a consequence, important therapeutic implications arise from a correct diagnosis of atypical presentations of mitochondrial disorders.In conclusion, mitochondrial gene mutations may present not only with different and less frequently atypical phenotypes, but they may also be considered as an adjunctive risk factor for ischemic events. In this case, we think that better knowledge of the genetic background could help not only in a correct diagnosis, but also for a tempestive and tailored therapy.Additional file 1. Supplementary materials. Family history and pedigree.


The past decades have witnessed advances in the development of ultrasensitive assays to measure biofluid markers to improve the diagnosis and characterization of central nervous system (CNS) involvement in primary and non-primary neurological diseases , 2. NeurRübsamen et al. investigated potential solutions for a better comparability of NfL concentrations measured in distinct biological matrices to harmonize findings across epidemiological studies. The authors confirmed the numerical differences between EDTA-plasma and serum NfL levels and derived a formula for converting NfL values from EDTA-plasma to serum, so that reference values for NfL standardization  can alsHavdal et al. investigated the relationship between neurological symptoms, cognitive performance , and blood biomarkers of brain injury  in a cohort of 405 adolescents and young adults (12–25 years) with paucisymptomatic COVID-19. They reported slightly higher levels of serum NfL and GFAP, while cognitive performance and clinical neurological examination did not differ compared to age-matched non-COVID-19 controls. Neurological symptoms such as fatigue, subjective cognitive difficulties, sleep alterations, and pain were not associated with increased serum NfL and GFAP levels nor with cognitive test results but showed a strong correlation with female sex and older age. These findings suggest a potential disconnection between self-reported neurological symptoms and biological abnormalities in mild COVID-19. Therefore, the authors argued that persisting clinical disturbances in these patients might rely more on biopsychological factors than ongoing underlying structural CNS damage.The role of NfL was highlighted in another study of the present issue. Huang et al. used an unsupervised hierarchical clustering analysis to outline a disease-specific CSF protein profile to improve the diagnosis of tuberculous meningitis (TBM). Specifically, a combination of 3 biomarkers  yielded the highest ability in discriminating TBM patients from cases with non-tubercular meningitis with an area under the curve (AUC) of 0.916 (95% sensitivity and 77.5% specificity). The higher expression of APOE proteins may reflect a dysregulation in CNS lipids metabolism directly caused by the pathogen. Whilst APOE proteins are mainly released by astrocytes, microglia, and neurons  in the CSF of 31 patients with aneurysmal subarachnoid hemorrhage (aSAH) using Olink® proteomic platform. A total of 15 proteins were upregulated in patients with PSBM compared to those without, and three proteins  yielded the highest diagnostic accuracy, thus outlining a potential distinct immunological profile of PSBM.Similarly, the study by He et al. evaluated the prognostic value of cellular elements evaluated in standard CSF cytology, particularly blood neutrophil-to-lymphocyte ratio (NTR) and monocyte-to-lymphocyte ratio (MLR) in patients with viral encephalitis (VE). The authors found significantly higher NTR and MLR at hospital admission in patients with poor  vs. good (mRS ≤ 1) prognosis at 12-month follow-up. Additionally, increased NTR and MLR were identified as independent predictors of 12-month poor functional outcomes. These findings might improve the early identification of critically ill VE patients who might benefit from more intensive monitoring and early treatment escalation to prevent poor prognosis.Finally, the study by Rübsamen et al. provides a possible solution to harmonize NfL levels when obtained in different biological matrices, thus potentially representing a significant step forward in the long road toward clinical validation.In conclusion, all the articles included in this Research Topic provide relevant advancements to implement CSF and blood markers in the diagnostic and prognostic assessment COVID-19 and other neuroinfectious diseases  and offeAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


This cohort study examines the association between the Affordable Care Act’s Medicare Advantage payment reductions and Medicare Advantage enrollment growth. Were the Affordable Care Act’s payment cuts to Medicare Advantage plans associated with differential reductions in Medicare Advantage enrollment?In this cohort study using a difference-in-differences analysis of 3138 counties with 37 639 county-year observations, during the 8 years following the Affordable Care Act, counties with larger cuts to Medicare Advantage plan payments had similar Medicare Advantage enrollment growth as counties facing smaller cuts.Payment cuts of the magnitude imposed by the Affordable Care Act did not appear to reduce enrollment growth in Medicare Advantage; modest plan payment cuts may reduce federal spending without compromising access to Medicare Advantage. Various policy proposals would reduce federal payments to Medicare Advantage (MA) plans. However, it is unclear whether payment reductions would compromise beneficiary access to the MA program.To quantify the association between MA payment reductions under the Affordable Care Act (ACA) and MA enrollment growth.This retrospective cohort study examined the MA market before and after the ACA, which mandated cuts to MA benchmark payment rates. Using 2008 to 2019 county-level enrollment and payment data, a difference-in-differences analysis was conducted comparing MA enrollment changes between counties with larger vs smaller benchmark reductions, before vs after the ACA.The primary outcome was the MA enrollment rate, defined as the proportion of a county’s Medicare beneficiaries enrolled in MA. A secondary analysis examined MA plan payments per member per month.P = .98). Plan payments differentially fell in counties with larger benchmark cuts by $78.35  per member per month (P < .001).Among 3138 counties with 37 639 county-year observations, ACA-induced benchmark cuts were sizeable and varied, ranging from 0% to 42.9% . Counties with benchmark cuts above the 75th percentile had population-weighted average benchmark cuts of 14.9% compared with 4.4% in other counties. In the 8 years following the ACA, there was no differential change in MA enrollment between counties with larger vs smaller benchmark cuts (difference-in-differences estimate, 0.02  percentage points; This cohort study found no evidence that the MA benchmark and ensuing payment cuts imposed by the ACA were associated with reduced MA enrollment, compromising access to MA. This evidence can inform ongoing policy debates regarding the growth of MA, concerns about excess payments to MA plans, and proposed Medicare reforms, including further reductions in MA payments. The MA risk-adjustment system, which scales MA plan payments based on enrollee comorbidities, has attracted particular criticism,3 prompting new expansions to federal oversight via audits.4 The Medicare Payment Advisory Commission estimates that payments to MA plans are several percentage points higher than the expected spending of MA beneficiaries had these beneficiaries enrolled in fee-for-service (FFS) Medicare.5 Greater payments to MA plans may allow MA plans to boost enrollment by offering more extensive benefit offerings than FFS Medicare. This association between the generosity of plan payments and MA enrollment has been documented in several studies that examined idiosyncratic variation in payment generosity.10Medicare Advantage (MA) is forecast to encompass half of overall Medicare enrollment by 2023, a new milestone in the vast expansion of private health insurance within Medicare.12 For example, MA appears to reduce hospitalization rates,15 without evidence of worsened mortality.16 Heart attack mortality had historically been lower in MA, though these mortality rates equalized over time.17 In addition, MA has also tended to perform well with respect to outpatient process measures of quality.11 Thus, it is possible that policy reforms to MA plan payment could reduce beneficiary access to valuable benefits.However, curbing MA plan payment could bring unintended consequences. The generosity of MA plan payments can finance supplemental benefits, such as premium reductions for prescription drug coverage, dental coverage, and gym memberships, and may promote health care quality. In quasi-experimental and descriptive studies, MA has generally performed as well or better than FFS Medicare on some measures of health care quality.5 served an important role as a source of financing for the ACA; in evaluating the budgetary influence of the legislation, the Congressional Budget Office projected that ACA reforms to MA would reduce federal spending by $136 billion from 2010 to 2019.18 The main policy mechanism for reducing MA plan payments was a revision to the methodology for calculating county benchmarks, a key element determining plan payment levels. Although these reforms did not appear to shrink the MA market immediately,19 little is known about their long-term consequences. Understanding the implications of payment cuts for MA growth could inform policy makers about the trade-off between promoting access to MA and containing governmental spending. In this retrospective cohort study, we examined the association between MA plan payment cuts from the ACA and MA enrollment.To inform this important debate, we studied a policy change in the Affordable Care Act (ACA) that reduced MA payments in different US counties to differing degrees. The ACA reforms to MA payment, arguably the most substantial MA reforms since the Medicare Modernization Act of 2003,22 We did not extend the analysis beyond 2019 to avoid data derived during the COVID-19 pandemic. We excluded a single county-year that reported greater than 100% MA enrollment. The research protocol was exempt from institutional review board review because it used only publicly available, aggregate data.This analysis used 2008 to 2019 Centers for Medicare & Medicaid Services’ public files, including MA ratebooks, MA state/county penetration files, and Part C plan payment files, which collectively contain county-level enrollment, benchmark, and payment data.Broadly, during the study period, 3 key factors determined the payments received by MA plans: (1) a county-level benchmark, (2) a plan-level bid, and (3) the characteristics of enrolled MA beneficiaries. Benchmarks reflect the maximum amount the government will pay an MA plan monthly for a typical beneficiary. Benchmarks are set based on FFS Medicare spending; the ACA modified the formula determining these benchmarks. Bids, which are submitted by plans, serve as the insurers’ price quotes for providing standard Part A and Part B benefits to a typical beneficiary. Bid levels and their difference from county benchmarks determine not only the plan payment level, but also whether enrollees must face an MA enrollment premium and whether the plan receives an additional rebate payment that can be used to finance additional MA benefits or reduce beneficiary Medicare premiums . Finally, plan payments are risk adjusted to account for beneficiary characteristics that are associated with higher or lower medical spending.23 Furthermore, a county’s post-ACA benchmark level was capped such that it could be no greater than a benchmark that would be calculated using the pre-ACA methodology. Consider the case of 2 LaSalle counties. In 2011, LaSalle, Illinois, and LaSalle, Texas, had respective benchmark levels of $737 and $1186 per beneficiary per month; in 2012, the respective post-ACA benchmarks for these counties were $759 and $730 per beneficiary per month. However, these benchmark changes were phased in over several years, during which time the effective benchmarks in each county were a blended mix of the pre-ACA and post-ACA benchmark calculations. All counties transitioned fully to ACA-set benchmarks by 2017. The reforms coincided with a reduction in average plan payments from approximately 112% of FFS spending in 2011 to 103% of FFS spending in 2019.5 See the eMethods in The ACA’s new MA payment formula included benchmark cuts targeted at counties with greater historical Medicare spending. The post-ACA benchmark formula introduced new adjustment factors based on a county’s quartile of per-capita FFS spending; counties with higher spending received benchmarks that were a lower fraction of spending.To quantify the magnitude of payment reductions, we calculated each county’s ACA-induced benchmark cut. We calculated this quantity as the difference between a county’s 2017 benchmark using the pre-ACA and post-ACA formula as a proportion of the pre-ACA 2017 benchmark. We examined 2017 benchmark changes because this was the first year that ACA benchmark calculations were fully in effect nationwide. Because 2017 benchmarks from the pre-ACA formula were included in the data source, we did not need to simulate counterfactual benchmark calculations. Because county benchmark changes depended on the county’s quartile of FFS spending, we grouped counties in the highest quartile of benchmark cuts in the primary analysis.The primary outcome, the county-level MA enrollment rate, was calculated yearly as MA enrollment divided by total Medicare enrollment for the month of June, which avoids the MA open enrollment period spanning January to March. For 960 county-year observations whose enrollment data were masked due to fewer than 10 MA enrollees, we imputed an MA enrollment rate of 0%.23 To visualize payment changes over time, we generated a weighted average of plan payments per member per month, weighted by estimates of plan enrollee count. See the eMethods in The secondary outcome was federal MA plan payment levels. The measure of plan payment includes not only base payment rates, but also risk adjustment for beneficiary characteristics and additional rebate payments. To focus the analysis on government payment generosity, we did not include beneficiary premiums in this measure. Because benchmark levels are one of several parameters determining plan payment, the ACA benchmark reforms did not guarantee that MA plan payments would fall in targeted counties. Payment levels might have been stabilized by counteracting actions of insurers, such as changes to bidding behavior, or by other features of the ACA reforms, such as a new quality bonus program.We conducted a difference-in-differences analysis to compare changes in MA enrollment and plan payments following the ACA for counties experiencing larger benchmark cuts (75th percentile or above) vs smaller benchmark cuts . We modeled the phase-in period of 2012 to 2016 as a washout period, assessing the post-ACA enrollment outcomes beginning in 2017 after the phase in of post-ACA benchmarks. Robust standard errors were clustered at the county level. We weighted county-year observations by Medicare population using the total number of county Medicare enrollees in 2011. In sensitivity analyses, we used alternate definitions of larger vs smaller benchmark cuts, used continuous measures of benchmark cuts, adjusted for any pre-ACA nonparallel temporal trends in outcome, considered the washout period as part of the post-ACA period, and conducted unweighted analyses.In the difference-in-differences analysis of the secondary outcome, plan payment levels, the unit of analysis was the county-year-plan type . See the eMethods in P < .05 considered statistically significant.All analyses were conducted in Stata, version 16.1 (StataCorp). All statistical tests were 2-sided, with The sample included 3138 counties and 37 639 county-year observations with a mean (SD) 2017 benchmark of $827.37 ($53.32) per beneficiary per month. The ACA-induced benchmark cuts were sizeable and varied, ranging from 0% to 42.9% . Sensitivity analyses, including models adjusting for any differential pre-ACA enrollment trends, also failed to detect a negative association between benchmark cuts and enrollment growth (Larger benchmark cuts were not associated with lesser growth in MA enrollment . The prit growth .P < .001). Again, sensitivity analyses yielded similar results  and that recent MA growth likely reflects a multitude of secular trends.This study did not find evidence of reductions in MA enrollment associated with the MA plan payment reforms of the ACA. In the 8 years after the policy change, this analysis confirmed that the counties most affected by the reforms, as measured by benchmark reductions, experienced relative reductions in plan payment levels compared with counties facing smaller benchmark cuts; however, they experienced similar MA enrollment growth. This study extends prior research showing no immediate effects of the ACA on MA enrollment in the 1 year after ACA reforms.4 One interpretation of these findings is that the MA program could absorb further payment cuts without shrinking the MA market. Indeed, the present results provide direct evidence that ACA-induced payment reductions did not curb MA enrollment. This finding also provides indirect evidence that the ACA payment reductions did not compromise benefits highly valued by Medicare beneficiaries who were deciding whether to enroll in MA. However, we cannot rule out the possibility that the ACA’s payment cuts reduced the value of MA plan offerings but that these changes did not affect overall MA enrollment. For example, MA insurers might have reduced the generosity of certain benefits that prospective enrollees undervalue (perhaps inappropriately) or that are valued only by inframarginal MA enrollees . Under these circumstances, Medicare beneficiaries could be worse off even without changes in MA enrollment. These possibilities should be investigated in further research examining changes in MA benefit offerings after the ACA. Finally, it is important to note that the enrollment effects of future MA payment reductions, even of the same magnitude, could differ from those observed following the ACA; if MA plan payments fell to a greater degree, then more striking reductions in benefit generosity and enrollment might occur. Also, payment cuts may have different effects depending on which aspect of the payment calculations is modified .This study can inform ongoing policy debates regarding the growth of MA, concerns about excess payments to MA plans, and proposed Medicare reforms including further changes to MA benchmarks.10 For example, several studies have examined the association between increased MA payments for urban counties and increased MA enrollment.9 These studies focused on quasi-experimental variation in plan payments arising from a 2003 policy regarding so-called urban floor counties; counties received higher MA plan payments if they were part of metropolitan statistical areas with populations of 250 000 or more and had relatively low FFS spending. Other research has demonstrated an association between MA plan payments and MA enrollment by examining variation in benchmark payments that were uncorrelated with county characteristics and local FFS spending.10This study’s key findings differ from prior research showing associations of idiosyncratic variation in MA benchmark levels with changes in MA enrollment.The divergence between the present findings and past research may reflect distinctions between the ACA reforms and previously studied sources of MA plan payment variation. For example, we studied payment reductions while others studied payment increases due to the urban floor feature of plan payment calculations. It is possible that insurers respond asymmetrically to payment cuts and payment increases, passing on the benefits of payment increases to consumers to attract enrollment but shielding existing benefits from the payment cuts. Insurers’ response to the ACA may have been muted by the timing of the reform, which occurred after many years of rapid national MA growth and featured a multiyear phase in of new benchmark rates. The ACA also targeted payment cuts to counties with higher benchmark levels and enrollment at baseline. Insurers in these markets may have been uniquely able to absorb payment cuts without compromising benefit generosity . These mechanisms could be elucidated in future research quantifying whether some insurers responded to ACA payment reforms via changes in benefit offerings, bid levels, or other behaviors, such as increased coding of beneficiary diagnoses, and whether any of these insurer responses prompted changes in beneficiary demand for MA plans.This study has several notable limitations. First, the difference-in-differences analysis required a central assumption that, in the absence of the ACA-induced payment cuts, MA enrollment would have changed over time similarly between the counties with larger benchmark reductions and other counties. We did not detect differential MA enrollment trends in the pre-ACA period, which suggests that enrollment trajectories were similar. Second, the results would be subject to bias if the ACA coincided with simultaneous changes in other factors that influenced MA enrollment differently in treatment and control counties. The ACA entailed a multitude of policy changes; however, we are not aware of other contemporaneous policy changes that would have affected MA enrollment differentially in counties facing payment cuts. Third, as discussed previously, it remains unclear why benchmark reductions were not associated with slowed MA enrollment growth. However, we confirmed that benchmark cuts were associated with substantial relative reductions in MA plan payment amounts. This suggests that the ACA benchmark reductions indeed reduced plan payments in a manner that was not reversed by MA plan responses to the ACA or other aspects of the ACA’s plan payment reforms.This retrospective cohort study found that the ACA’s MA payment reforms were not associated with reductions in MA enrollment. Counties that faced greater ACA-induced benchmark reductions, and therefore greater cuts to MA plan payments, experienced similar MA enrollment growth as other counties. In the context of the ongoing debate regarding the size and federal costs of the MA program, these findings suggest that prior reforms were associated with lower federal spending on MA without notably compromising beneficiary access to the MA market. Further research could illustrate what features of the MA marketplace prevented these payment cuts from curbing enrollment growth and investigate if consumer welfare was affected in other ways.


Portal vein thrombosis (PVT) has been usually diagnosed as a complication secondary to cardiac, hepatic, and malignant etiologies, but it has rarely been described in the setting of blunt abdominal trauma. This case depicts an older male who presented to the emergency department with progressive ascites and lower extremity edema within two weeks after a motor vehicle accident (MVA). Ascitic fluid analysis indicated the presence of portal hypertension, which prompted extensive evaluation to determine the etiology. Further workup, including hypercoagulable and malignancy screening, unveiled the diagnosis of acute PVT secondary to abdominal blunt force trauma, showing a rather rare presentation. Portal vein thrombosis (PVT) is the narrowing or total occlusion of the portal vein secondary to a thrombus and accounts for approximately 5-20% of the cases of portal hypertension ,2. CommoThis case portrays an acute portal venous thrombosis as a rare complication following blunt abdominal trauma. We present a case of a patient with new-onset ascites and abdominal pain associated with PVT two weeks after a motor vehicle accident (MVA).A 62-year-old male with hypertension, diabetes mellitus type II, and chronic kidney disease presented to the emergency department with sharp right upper quadrant (RUQ) abdominal pain radiating to the right lower quadrant associated with bilateral lower extremity edema. Symptoms started two weeks prior to an MVA and had progressed since onset. The patient was hemodynamically stable and a febrile. Physical examination was remarkable for mild RUQ abdominal tenderness, distended abdomen with fluid wave present, and grade IV bilateral lower extremity edema. Remarkable laboratories are demonstrated in Table Laboratories were unremarkable for leukocytosis, thrombocytopenia, abnormal coagulation studies, electrolyte abnormalities, negative smooth muscle antibodies, and mitochondrial antibodies. D-dimer was not obtained in the initial presentation. Computed tomography (CT) of the abdomen and pelvis without contrast was remarkable for bilateral pleural effusions, the right being greater than the left; gallbladder thickening and pericholecystic edema; thickening and edema in the region of descending duodenum wall; and abdominal ascites Figure . AbdomenThe most common symptoms among non-malignant and non-cirrhotic acute PVT cases are abdominal pain up to 91%), followed by fever 53%) and ascites 38%) 3% and as,7. The c%, follow% 3% and PVT is a rare occurrence in a rather healthy person without risk factors. Oftentimes, symptoms are broad and non-specific, which makes its diagnosis even more challenging. This case depicts a rare presentation of ascites in a non-cirrhotic patient with acute PVT in the setting of blunt abdominal trauma without acute complications. We aim to highlight the importance of accurate diagnosis, treatment, and management of atypical acute cases, such as our patient in a multi-disciplinary approach that will decrease the risk of long-term complications, improve survival rate, and optimize their quality of life.


Fluorescent labelingallows for imaging and trackingof vesiclesdown to single-particle level. Among several options to introducefluorescence, staining of lipid membranes with lipophilic dyes providesa straightforward approach without interfering with vesicle content.However, incorporating lipophilic molecules into vesicle membranesin an aqueous solution is generally not efficient because of theirlow water solubility. Here, we describe a simple, fast (<30 min),and highly effective procedure for fluorescent labeling of vesiclesincluding natural extracellular vesicles. By adjusting the ionic strengthof the staining buffer with NaCl, the aggregation status of DiI, arepresentative lipophilic tracer, can be controlled reversibly. Usingcell-derived vesicles as a model system, we show that dispersion ofDiI under low-salt condition improved its incorporation into vesiclesby a factor of 290. In addition, increasing NaCl concentration afterlabeling induced free dye molecules to form aggregates, which canbe filtered and thus effectively removed without ultracentrifugation.We consistently observed 6- to 85-fold increases in the labeled vesiclecount across different types of dyes and vesicles. The method is expectedto reduce the concern about off-target labeling resulting from theuse of high concentrations of dyes. Water-soluble dyes behave largelyindependent of the vesicle content, but cannot be internalized intopreformed vesicles such as purified EVs due to the membrane barrier.Although some nonfluorescent, membrane-permeant molecules, such ascarboxyfluorescein diacetate succinimidyl ester (CFDA-SE), can passivelydiffuse into vesicles and then become fluorescent,3 they only work with vesicles containing active esterasesand therefore will be biased by the vesicle content. Membrane stainingwith lipophilic tracers offers unbiased and bright labeling: a varietyof cyanine-derivative dyes with single-molecule sensitivity are developedacross the entire spectrum of visible light.10 Since a typical, 100 nm vesicle carries ∼80000 lipid molecules,introducing only 0.01 mol % of lipophilic dyes will yield ∼8dye molecules per vesicle on average, sufficient for single-particletracking. Additionally, these molecules naturally exhibit a largeincrease in fluorescence upon partitioning into the membrane, furthercontributing to the high signal-to-noise ratio against free dye molecules.Several methods to fluorescently label EVs areavailable: immunostainingof surface proteins, internal protein tagging with membrane-permeabledyes, use of water-soluble dyes inside vesicles, or membrane stainingwith lipophilic dyes.13 These problems are associated with the use of highconcentrations of dyes that are used to force the incorporation oflipophilic molecules into closed, preformed vesicles. Although slightlysoluble in water, most of the lipophilic molecules are kineticallytrapped in large aggregates, so the passage from aggregates to membranesis unfavorable. One can drive the equilibrium toward vesicles by increasingdye concentrations, but at the expense of additional sample processingto remove the large amount of free dyes, which is frequently accompaniedby severe loss of vesicles. Moreover, a recent work suggests thatthe incorporation of large dye aggregates may also lead to a significantincrease in vesicle size.14 Consequently,an effective method to fluorescently label vesicles with high sensitivity,high specificity, and simplicity to preserve the quality and quantityof vesicle samples is desirable.Notably, however, vesicle staining with lipophilic dyes often suffersfrom confounding factors, such as the complications from off-targetlabeling of lipoproteins and free-dye aggregates that are similarin size to EVs.Here, we describe an improvedmethod for the fluorescent labelingof vesicle samples, including EVs. This method is simple, fast, andeffective both in improving the labeling efficiency and in removingunwanted free dyes. By characterizing the aggregation status of alipophilic tracer DiI 1 as a fuSupporting Information. For the salt-change labeling, vesicle samples in PBS were dilutedto 1010 particles/mL with ultrapure water to lower NaClconcentration to below 20 mM and reacted with 2 μM DiI or DiDfor 20 min at 37 °C. A small volume of PBS concentrate (10×)was then added to increase NaCl concentration to ∼150 mM. Fordirect staining, the same concentration of dyes was directly appliedto vesicles in regular PBS with 150 mM NaCl. The mixture was filteredthrough a 0.2 μm syringe filter  to removeaggregates of dye molecules and subjected to further experiments.Labeling results were verified by imaging of either free-floatingor anti-CD63-bound vesicles on a TIRF microscope (see Video S1 for the representative results) as describedin the Supporting Information.Preparation of vesiclesare described in the P values for the octanol/water partition coefficient15 are given in 16 . After introducing DiI solutions into a glass flow cell,the fluorescent particles floating by near the glass surface wereilluminated.Although lipophilicdyes are commercially available as powders or as solutions in organicsolvents such as ethanol, dimethylformamide (DMF), or dimethyl sulfoxide(DMSO), they need to be transferred to aqueous solutions for vesiclelabeling not to disrupt vesicles and the embedded proteins. This requirementposes a challenge because the water solubility of lipophilic moleculesis generally low, as shown by their high lipophilicity , whichare likely large aggregates of DiI rather than single molecules. Sincemost fluorescent dyes including DiI exhibits aggregation-caused quenchingof fluorescence intensity, the brightness of the particles would actuallyunderestimate the number of dye molecules per particle. Indeed, theseaggregates were completely removed by filtering the solution through0.2 μm pores , implyingthat they are mostly micron-sized. These large aggregates are likelyinefficient at labeling vesicular membranes and may cause an increasein vesicle size after labeling.14We first imaged 2 μM DiI solution in a bufferwith ∼150 mM NaCl, a physiological and typical condition forcommon buffers including PBS. A small number of bright, slowly movingparticles were detected . Importantly, we checked that these changes to particles occurredwhile the total amount of dye molecules and their fluorescence remainedconstant: After solubilizing the dye aggregates completely with detergent(0.1% Triton X-100), the overall fluorescence intensity from the solutionwas measured to be the same across the concentrations of NaCl we tested. In contrast, the fluorescencefrom the buffer with 155 mM NaCl almost completely disappeared aftermicropore filtering , suggesting thatmost of the dye molecules in this condition were trapped in the aggregatesand subsequently removed.We therefore attempted to improve the solubilization of DiI bydecreasing NaCl concentration. The aggregates gradually dispersedas NaCl concentration was lowered to ∼20 mM, as shown by theincrease of relatively dim particles  were prepared by using a published procedure,18 and labeled them with DiI ), a lipophilic fluorescent tracer for labelinglipid membranes.The above results suggest that dispersionof DiI in a buffer witha low concentration of NaCl can potentiate membrane partitioning ofDiI and that the excess dye can be removed by inducing its aggregationat a higher concentration of NaCl. We therefore exploited this reversibleaggregation of DiI to improve the labeling of vesicles 2. To ver16  “Direct staining”performed with 150 mM NaCl, that is, in regular PBS 2A, and 2B. In th16 2C.Video S1). Since the CDVswere prepared at a fairly high concentration (∼1010 particles/mL), we expected much more particles to be present inthe field of view, and therefore, it was very unlikely that all CDVswere successfully labeled by the direct staining method. Althoughthe observed level of labeling efficiency might be suitable for bulkassays that probe many vesicles at the same time , the labeled CDVs were neither sufficiently abundantnor sufficiently bright for quantitative measurements at the single-vesiclelevel. According to our observations of NaCl concentration-dependentDiI aggregation, we argued that the low labeling efficiency wouldstem from poor solubilization of lipophilic dyes in the staining buffer.19 In stark contrast, the salt-change method increasedthe number of bright fluorescent vesicles 85 ± 10 times (Video S1), and their average brightness alsoincreased 2.3 times compared to vesicles stained in PBS with 150 mMNaCl . We estimated the number of DiI molecules in each vesiclefrom the ratio of the initial fluorescence to photobleaching stepsize . Each CDV typicallycarried 1–3 molecules of DiI, and these numbers followed aPoisson distribution as expected . The results imply that only a small fraction of the CDVs remainedunlabeled and, at the same time, that the mole fraction of DiI invesicle membranes was <10–4 .Therefore, the labeling density we achieved was sufficient for single-vesicleimaging, but unlikely to disrupt the native properties of the membrane.To accurately measurethe labeling density , the labeled NK-CDVs were stably captured ona surface and their fluorescence intensity was measured , consistent with the results forNK-CDVs. It is remarkable that the proposed method was much more effectivethan adding dimethyl sulfoxide (DMSO) to the staining buffer , a common approach to improve thesolubility of lipophilic dyes. Also, if the syringe filters for dyeremoval were not rinsed with buffer (2 mL of PBS) before use, we noticedthat the vesicle yield decreased slightly , possibly due to the trace wetting agents inthe off-the-shelf cellulose acetate filter membranes.To test whetherthe salt-change labeling method can be applied to other vesicles,we first prepared another sample of CDVs from umbilical cord mesenchymalstem cells (UCMSC-CDVs) and labeled them with DiI. Again, the salt-changemethod showed a dramatic improvement in labeling efficiency (Escherichia coli W3110 (a widelyused wild-type strain) (“E. coli OMV”),following a published procedure.21 Applying thesalt-change labeling to these vesicles, we again obtained a greatimprovement in labeling efficiency over direct staining in a high-saltbuffer ; 22 tail. It is thereforeexpected to be highly lipophilic, and presumably, the labeling strategyproved successful similarly to DiI and DiD. Together, these resultsdemonstrate the broad applicability of the salt-change method forthe fluorescent labeling of preformed vesicles with lipophilic dyes.Experiments with two other lipophilicdyes DiD, thus, the size difference between vesicles and free-dye aggregatescould be successfully exploited to purify labeled vesicles.Consistentwith the small amount of dye molecules per vesicle, the size distributionof NK-CDVs, as measured by nanoparticle tracking analysis (mediandiameter of ∼150 nm), was not distorted by the salt-changelabeling 4A.AlthoE. coli OMVs, and synthetic liposomes),too, but the yield somewhat depended on vesicle type . Theincrease in OMV number can be rationalized by the suboptimal detectionof small particles in NTA measurements, which became detected uponlabeling. Notably, although the recovery of NK-EV was relatively lowwhen 2 μM DiI was used, the yield was completely restored byusing 0.2–0.67 μM DiI (Video S2), suggestingthat an optimal dye concentration needs to be determined empiricallyfor a given sample of vesicles.Vesicle sizes were largely maintained for the othertypes of vesicles-coated glass surface with anti-CD63; and therecovery of input vesicles was satisfactory  and can be finished within 30 min in a regular wet lab.Improving the labeling efficiency also allows the use of lower concentrationsof dyes and vesicles and thereby reduce the possibility of nonspecificlabeling. We expect this method to apply to clinically obtained EVsfrom liquid biopsy, which will particularly benefit from effectivelabeling because they are usually limited in amount.The two-orders-of-magnitude improvementin labeling efficiencywith the salt-change method is impressive given its simple steps,and therefore can potentially substitute complex labeling and purificationprotocols (such as the standard protocol for PKH67 we used for comparison),even for researchers with access to an ultracentrifugation system.Figure S3C,D), butthe results will likely depend on the specific filter models in use.One concernwith the salt-change labeling is that ionic strengthchange may induce osmotic stress on vesicles and membrane proteins.We verified that the selected proteins (endogenous CD63 and overexpressedPD-1) were still recognized by antibodies after labeling 5. AlthouIn this study, we investigated fluorescentlabeling of vesicleswith lipophilic dyes, revealing a critical dependence on NaCl concentration.We exploited the reversible aggregation of DiI molecules both to improvelabeling efficiency and to remove free dye molecules from the vesiclesolution. The salt-change labeling method was shown to be widely applicableto many types of vesicles and dyes, without noticeably degrading thefunctional properties of vesicle samples and content proteins. Weexpect that this protocol will be useful in a broad spectrum of fluorescence-basedassays interrogating natural EVs and engineered nanovesicles.


Membrane-less organelles (MLOs) formed through liquid-liquid phase separation (LLPS) are associated with numerous important biological functions, but the abnormal phase separation will also dysregulate the physiological processes. Emerging evidence points to the importance of LLPS in human health and diseases. Nevertheless, despite recent advancements, our knowledge of the molecular relationship between LLPS and diseases is frequently incomplete. In this review, we outline our current understanding about how aberrant LLPS affects developmental disorders, tandem repeat disorders, cancers and viral infection. We also examine disease mechanisms driven by aberrant condensates, and highlight potential treatment approaches. This study seeks to expand our understanding of LLPS by providing a valuable new paradigm for understanding phase separation and human disorders, as well as to further translate our current knowledge regarding LLPS into therapeutic discoveries. Membrane-less organelles (MLOs) formed through liquid-liquid phase separation (LLPS), also known as biomolecular condensates, participate in various biological processes and are crucial for human health ,2 . TheDevelopmental disorders are diseases that entail deviations from normal development and often manifest during early childhood, of which, intellectual disability (ID) and autism are two examples. R168X mutant mouse embryonic stem cells also present evidence of Rett syndrome-associated cellular phenotypesTOP2A andTOP2B)Mutations result in decreased or eliminated protein LLPS capacity, which in turn leads to biological processes that are defective. R168X Furthermore, condensate disruption during neuronal development can potentially have an impact on synaptic distribution and neuronal identity. For example, the transcription factor Tbx3 plays a critical role in establishing and maintaining the identity of KNDy neurons, which trigger pubertyPTPN11, plays a crucial role in normal development by mediating RAS-mitogen-activated protein kinase (MAPK) signalingR636S granules significantly accumulate in the sarcoplasm and distribute on the myofibril Z-discsR636S granule may result in the sequestration of actin alpha cardiac muscle 1, further disrupting the actin cytoskeleton of cardiomyocytes [Gain of function denotes the possibility of activating or improving a protein’s capacity for phase separation as a result of mutations. Developmental diseases can be caused by the gain of LLPS, which result in disease-related pathways or reactions being hyperactivated or lead to the sequestration of RNAs, proteins, or both. The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded byyocytes ,50 .Different from the gain of function, protein gain of toxicity represents the mutation of the gene results in the acquisition of the aggregation propensity that can interrupt the normal function of the wild-type protein, which may lead to abnormal aggregation and cell toxicity. Similar to that in neurodegenerative diseases, the aggregation state of associated proteins is correlated with the dysregulation of physiological functionIn addition to developmental disorders, phase separation also plays a critical role in tandem repeat disorders (TRDs). TRDs are caused by the abnormal expansions of short tandem repeats (STRs) that are 2‒12 base pairs long DNA repeating tracts and locate in both coding and noncoding regions. More than 50 human disorders are now known to be caused by STR expansion ,55 . ThFMR1 gene can generate toxic RNA foci that are regulated by multivalent interactions, leading to intranuclear inclusion formation in fragile X-associated tremor and ataxia syndrome (FXTAS)HTT) gene promote a solid-like state and cause intracellular aggregation of HTT protein in Huntington’s disease (HD)A growing number of studies have discovered that aberrant phase separation is associated with gain-of-toxicity in STRs. For example, at RNA level, the premutation range of CGG repeat expansions in the 5′ UTR ofC9orf72 locus is the most frequently known cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) (in vitro andin vivo [in vitro. The longer length of these expanded RNA repeats promotes the formation of gel-like or solid-like dropletsDMPK) gene [The formation of RNA foci is a classic example of an RNA-mediated gain of toxicity mechanism. This process is intricate and involves the formation of unusual secondary structures by repeat RNA products. Through Watson-Crick and noncanonical base pairing, repeat RNAs can form imperfect hairpin structures and G-quadruplexes. The GGGGCC expansion in the intron ofLS/FTD) . GGGGCC in vivo ‒79 . ReK) gene ‒82 (FiK) gene ,84 .FDMPK RNA foci in the cell nucleus recruit a multi-functional RBP—muscleblind-like splicing regulator 1 (MBNL1). The expandedDMPK RNA and MBNL1 are regulators of the formation and turnover of cytoplasmic SGs in DM1CNBP) gene. The expandedCNBP RNAs and multi-functional RBP MBNL1 promote the assembly of ribonucleoprotein (RNP) granules or RNA foci in DM2FMR1 [FMR1 RNA. The RBPs complex includes DiGeorge syndrome critical region gene 8 (DGCR8), heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and src-associated substrate during mitosis of 68 kDa (Sam68). The RNA foci behave as ribonucleoprotein (RNP) condensates that phase separate in the nucleus, forming large ubiquitylated inclusionsBesides repeat RNAs, RNA-binding proteins (RBPs) attracted by expanded repeat RNAs can also impair the dynamics of condensates and stabilize the formation of RNA fociM2FMR1 ‒88 -containing proteins, which in turn results in impaired functions of multiple membrane-less organellesFMR1 mRNA containing expanded CGG repeats initiates RAN translation and produces a polyglycine-containing protein, FMRpolyG  translation of toxic peptides contributes to the pathogenesis of various TRDs. RAN translation represents the translation of tandem repeats into peptides consisting of repeating amino acid sequences that do not require AUG initiationroducts ‒93 . ReMRpolyG . The polWhen the tandem repeats locate within an exon coding region, the resulting TRDs typically exhibit gain-of-function phenotypes, characterized by abnormal protein aggregation and phase separation ,99 . InAt least nine disorders, including Huntington disease (HD), several spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA), are caused by CAG repeat expansions in coding sequences that result in the expressions of polyglutamine-containing proteinsHTT gene, while HD patients contain 36–121 repeats  protein. Wild-type individuals contain 6–34 CAG repeats in therepeats . The expAnother main type of TRD with tandem repeated polyglutamine tracts in an exon is Spinocerebellar ataxias (SCAs). The clinical hallmark of all SCAs is progressive atrophy of the cerebellum, brainstem, and spinal cordThe repeat expansions in an exon that encodes polyalanine tracts instead of the polyglutamine tracts outlined above are frequently linked to severe developmental abnormalities like synpolydactyly, X-linked mental retardation, and muscular dystrophyThe polyalanine tracts in IDR of these three typical TFs  show varied effects on their phase separation ability, including both impairment and enhancement. Firstly, HOXA13 is a homeobox TF. The polyalanine repeat expansion from 18 to 24–26 in the N-terminal IDR of HOXA13 results in hand-foot-genital syndrome (HFGS)In addition to transcription factors (TFs), alanine repeat expansions also occur in polyA binding proteins. PABPN1 is an abundant nuclear protein that binds with high affinity to nascent polyA tails. The RNA binding protein PABPN1 promotes the formation of nuclear polyA domains (NPADs) through its N-terminal disordered domain and RNA-recognized motif by liquid phase separationNOTCH2NLC (N2C) gene causes neuronal intranuclear inclusion disease (NIID), which is a neurodegenerative disease characterized by the presence of intranuclear inclusionsIn addition to polyglutamine and polyalanine disease, polyglycine (polyG) disease is defined as a novel class of TRDs recently. Expansion of GGC repeats in the 5′ UTR of theAltogether, short tandem repeats may alter the structure and function of proteins, differentially engage with their interacting partners and result in abnormal phase separation in the form of intracellular inclusions, and finally lead to neurological and muscular disorders cordIn addition to the tandem repeat disorders and developmental disorders, aberrant MLOs have also been linked to a range of cancers by disrupting tumor suppression and normal signal transduction pathways, hyperactivating oncogenic genes or affecting protein quality control machinery. In this part, we will discuss tumorigenesis in relation to abnormal phase separation .TableCondensates are essential in preventing and restraining cancer development. They achieve this by assembling protein complexes involved in tumor suppression, and enhancing the expression of the related gene to suppress antitumor immune surveillance. However, the abnormal condensates will impair these tumor-suppressive functions. For instance, promyelocytic leukemia (PML) bodies are submicron-scale nuclear membrane-less organelles composed of proteins including the PML protein which function as tumor suppressors ,137 . PAbnormal condensates exert a significant influence on transcriptional processes, thereby driving the development and progression of cancer. These aberrant biomolecular assemblies can upregulate the expression of oncogenes, leading to cancer cell growth and survival. For example, ENL is a histone acetylation reader that uses its YEATS (Yes-associated protein) domain to recognize acetylated lysine residuesIn addition to dysregulated gene expression, abnormal MLOs can also affect signal transduction related to cancer. Recent papers have shown that mutant protein-formed condensates result in abnormal signal transduction and contribute to tumorigenesis. One such example is the non-receptor protein tyrosine phosphatase SHP2, which was mentioned above in Noonan syndrome and Leopard syndrome. Certain mutations result in hyperactivated RAS-MAPK pathway by activating SHP2 enzymatic activity or by recruiting and activating wild-type SHP2 protein, and ultimately cause malignancies ,47 . AdApart from influencing transcription and signal transduction, mutations in proteins may impact the formation of condensates that are associated with protein degradation, potentially contributing to the development of cancer. SPOP (speckle-type POZ protein) is a protein that belongs to a family of enzymes called E3 ubiquitin ligasesAbnormal protein phase separation is closely associated with several non-communicable diseases. Recent studies, however, have shown that protein phase separation also plays a significant role in the emergence of some infectious diseases, particularly viral disorders. For most viruses, phase separation is involved in the majority of viral lifecycles, including protein synthesis, genome assembly, virus assembly, budding and release. For example, the 52-KDa protein of human adenovirus regulates viral assembly by phase separation, and failure to form condensates results in failed packaging and assembly of only non-infectious particles. In contrast to non-communicable diseases, LLPS in viral diseases occurs in two distinct ways. It can either be employed by the host as a defense mechanism against invading pathogens, or it can be utilized by the pathogen to aid in their invasion ‒159 .Innate immunity is a non-specific immune response and is also the first line of defense against pathogens. Upon recognizing the pathogen, host cells initiate a cascade of events to activate the cells to attack and kill the pathogens. The cyclic GMP-AMP synthase  is an enzyme that initiates the innate immune response after detecting double-stranded DNA (dsDNA), either from the pathogen or self, in the cytoplasm ,161 . ISGs are considered as a defense mechanism against various stresses, which can sequester certain transcripts and proteins from the soluble portion of cytoplasm during physiological stress ,169 . IPhase separation is a process that has been exploited by viruses. In addition to countering host condensate-based antiviral cell signaling, viruses have developed various strategies to take advantage of condensates enriched with both host and viral proteins for their replication, assembly and trafficking . During Occasionally, phase separation plays a dual role during viral infections, serving both advantageous and disadvantageous functions depending on the specific situations and interactions at play. On one hand, phase separation is beneficial for the virus infection process. The p26 movement protein from the Pea enation mosaic virus 2 (PEMV2) undergoes phase separation and forms droplets within the host cellIn recent years, it has been discovered that concentrating only on individual molecules may not be able to fully explain the complex illness phenotypes, and that it is also challenging to make a beneficial contribution to disease treatment methods by changing the structure or function of a single protein or nucleic acid. The investigation of phase separation may provide complex disease mechanisms, and even open up exciting new avenues for therapeutic intervention. We have discussed in detail how abnormal phase separation can lead to developmental abnormalities, neurodegenerative diseases, cancers, and viral infection. However, in most occasions, the relationships between phase separation and pathogenesis are currently simply correlative rather than causative, and warrant further investigation. In the meantime, finding effective therapies for diseases caused by abnormal phase separation remain challenging. Here, we list several potential therapeutic strategies. (1) Develop small molecules that can change the conformation of mutant proteins to inhibit their abnormal phase separation. For example, the allosteric inhibitors of SHP2 attenuate the phase separation of mutant SHP2 by locking it in a closed conformationIn this review, we have laid out that phase separation is significantly correlated with various diseases and showed that both normal and abnormal phase separation can be related to disease onset. Aberrantly disrupted phase separation causes transcription dysregulation, chromatin architecture changes, low ubiquitination activity or impaired innate immunity. The mutants that gain LLPS ability can promote the condensate formation and lead to hyperactivating the disease-associated pathway or overexpressing the oncogenic genes. In addition, the fibrillar structure or the liquid-to-solid transition, which allows for the conversion into solid-like states, is the primary cause of many neurodegenerative illnesses. Further evidence that phase separation may be the primary cause of diseases is provided by the fact that LLPS-associated diseases are broadly spread across the entire human body, as illustrated in


Ultrasound is a crucial and effective diagnostic tool in medicine. Recent advancements in technology have led to increased use of point-of-care ultrasound (POCUS). Access to ultrasound equipment and training programs in low-and middle-income countries (LMICs) is limited. Despite the World Health Organization (WHO) recommendations for universal antenatal ultrasounds, POCUS for reproductive health applications has not been widely used in LMICs. We describe here the feasibility of implementation of a training of obstetrics point-of-care ultrasound (OB POCUS) for high-risk conditions in rural public healthcare facilities in Kenya with partnership from Butterfly Network, Global Ultrasound Institute, and Kenyatta University.As part of the initiation of a large-scale implementation study of OB POCUS, clinician trainees were recruited from rural Kenyan hospitals for participation in a series of five-day POCUS workshops held between September and December 2022. Trainers provided brief didactic lessons followed by hands-on training with live models and at regional clinical sites for 5 OB POCUS applications. Instructor-observed assessment of students’ scanning and image interpretation occurred over the training period. Assessment of knowledge and confidence was performed via an online pre-test and post-test as well as Objective Structured Clinical Examination (OSCE) was administered at course completion.Five hundred and fourteen mid-level Health Care Providers (HCPs) in Kenya were trained over a three-month period through in-person didactic sessions, bedside instruction, and clinical practice over a 5-day period with a trainer: trainee ratio of approximately 1:5. Out of the 514 trained HCPs, 468 were from 8 rural counties with poor maternal and neonatal outcomes, while the remaining 46 were from nearby facilities. OB POCUS topics covered included: malpresentation, multiple gestation, fetal cardiac activity, abnormalities of the placenta and amniotic fluid volume. There was marked improvement in the post training test scores compared to the pretest scores.Our implementation description serves as a guide for successful rapid dissemination of OB POCUS training for mid-level providers. Our experience demonstrates the feasibility of a short intensive POCUS training to rapidly establish specific POCUS skills in efforts to rapidly scale POCUS access and services. There is a widespread need for expanding access to ultrasound in pregnancy through accessible OB POCUS training programs. An implementation study is currently underway to assess the patient and systems-level impact of the training.The online version contains supplementary material available at 10.1186/s12909-023-04886-x. Patient access to ultrasound in low- and middle-income countries (LMICs) remains a major obstacle, compromising clinician decision-making and patient outcomes . Point-oThere is substantial evidence to support the clinical value of POCUS to several specialties –5. For iPOCUS is highly operator-dependent , and musWhilst several national and international bodies have developed specialty-specific POCUS curricula, at the time of the training, there had not yet been an approved obstetrics POCUS (OB POCUS) curriculum in Kenya –10. POCUOur training initiative was nested in a larger ongoing study on the impact of OB POCUS on patient outcomes, health systems, and policy and is a collaboration with three partners: 1) Global Ultrasound Institute (GUSI), which served as the training partner; (2) Butterfly Network, the ultrasound solution; and (3) Kenyatta University (KU), which served as the research and recruiting partner. Out of the 514 HCPs who were trained, 468 were from 8 rural counties with poor maternal and neonatal outcomes [ Global UAs part of the Butterfly probe distribution project, the Kenyan team embarked on training 514 HCPs in OB POCUS skills specifically for pregnant patients in the second and third trimesters. This entailed knowledge and skills acquisition in five key thematic areas namely: overview scan for multiple gestation, fetal presentation and lie, placental location, amniotic fluid assessment, and fetal heart rate assessment. The project aimed to train the 514 HCPs over a period of 10 weeks. The learners were trained in groups of 50 HCPs every 5 days, with 10 instructors every day. Learners were issued Butterfly iQ + ™ hand-held ultrasound probes and Apple iPads loaded with just-in time education which they used during the training and later at their local work sites.Kenyatta University selected 8 out of the 10 high priority counties as identified by USAID to have high maternal and neonatal morbidity and mortality rates . These cOB POCUS instructors were selected from various disciplines in the medical field. These included sonographers, resident physicians in obstetrics and gynecology and family medicine, as well as an emergency physician. All had some level of ultrasound experience prior to the project.Instructor education sessions, entitled, “Lunch and learn sessions,” were held on 3 occasions. During each, master POCUS instructors taught the program’s instructors the course content as well as effective methods in POCUS education. OB POCUS topics in the course content adhered to the International Society for Ultrasound in Obstetrics and Gynecology (ISUOG) standard ultrasound techniques . In addiPrior to each workshop, trainees completed a knowledge pre-test  attained the pass mark of 13 out of 26. The mean test score was 87.3% - demonstrating a good grasp in obstetric POCUS skills. Learners who did not attain the pass mark were allowed to retake the OSCE and passed on the second attempt.Ultrasound is an extremely useful diagnostic imaging modality. It is noninvasive, provides real-time information, and is completely safe since it has no radiation exposure . The useThe current project employed a structured 5-day training program for HCPs encompassing online resources, didactic lecture sessions, live demonstrations, practice opportunities in various hospitals, assessment and a system for remote post-course support. Although effective instruction in the short training time available remains a challenge, it can be quite effective if used optimally. Fortunately, recent data suggest that those keen to learn POCUS can obtain adequate proficiency with minimal training, including online web-based training , 9, 16.During the training, it was emphasized that the aim of POCUS was to answer specific clinical questions that aid clinical decision-making to optimize patient outcomes –18. POCUThe current project trained HCPs currently involved in maternal care such as nurses, midwives, clinical officers, medical officers, radiographers and sonographers. Since POCUS is inconsistently taught in medical school , 10, sevJarwan et al. (2020) from Saudi Arabia assessed the needs and barriers to POCUS training among medical interns. They noted that POCUS is applicable to medical interns but significant skill gaps existed, including lack of time for training. They concluded that prioritizing the training of residents in POCUS would be a more effective use of the finite resources available for medical education instead of training medical interns who were mainly keen on medical licensing and applying for residency .This study is part of a larger funded initiative to improve diagnostic imaging access in order to propel Kenya towards achieving improvements in their maternal health outcomes. To meet the WHO recommendations for antenatal imaging, health departments should prioritize the acquisition of hand-held ultrasound machines paired with clinician training, which may serve to reduce patient barriers to necessary medical imaging. Making bedside imaging available to critically ill patients and those that require imaging outside of normal business hours will allow clinicians to make life-saving diagnoses of high-risk conditions in their most critical patients , 3. The The study has potential limitations. The assessment multiple choice questions had not been standardized nor validated in any other study or setting. They were developed by the authors and therefore may require reframing and validation in other training for a more widespread application.We report the experience of training 514 HCPs in obstetric POCUS using didactic lectures and hands-on experience over a 5-day period using the ISUOG standard ultrasound techniques. Follow up data will be collected to assess the impact of the current training and deployment of portable ultrasound devices to mid-level HCPs in hard-to-reach areas with little or no access to ultrasound services.Larger, more widespread OB POCUS training programs and estimation of their clinical impact are needed in LMIs. More broadly, the development of widespread POCUS training curricula for a wide variety of clinical applications should be embedded as an integral component of medical education in these settings. We encourage the integration of ultrasound training in medical schools throughout LMICs, which could have enormous potential impact on patient care in the future.Additional file 1. Pre and Post Test. Pre and Post Test used to assess baseline knowledge and gained knowledge of learners.Additional file 2. Logbook for Basic Obstetric Ultrasound. Learner’s logbook for tracking and recording ultrasound scans completed during the training.Additional file 3. OSCE Evaluation Form. Observed Structured Clinical Exam (OSCE) used to evaluate learners on the final day of training.


Emergency department (ED) visits for conditions unrelated to the Coronavirus Disease 2019 (COVID-19) pandemic decreased during the early pandemic, raising concerns about critically ill patients forgoing care and increasing their risk of adverse outcomes. It is unclear if Hispanic and Black adults, who have a high prevalence of chronic conditions, sought medical assistance for acute emergencies during this time. This study used 2018–2020 ED visit data from the largest safety net hospital in Los Angeles County to estimate ED visit differences for cardiac emergencies, diabetic complications, and strokes, during the first societal lockdown among Black and Hispanic patients using time series analyses. Emergency department visits were lower than the expected levels during the first societal lockdown. However, after the lockdown ended, Black patients experienced a rebound in ED visits while visits for Hispanics remained depressed. Future research could identify barriers Hispanics experienced that contributed to prolonged ED avoidance. During the earliest days of the Coronavirus Disease 2019 (COVID-19) pandemic, United States (US) healthcare centers saw a dramatic reduction in emergency department (ED) visits for non-COVID-19 care . SeveralAs many acute medical emergencies are the result of untreated chronic disease, it is unlikely the pandemic reduced chronic disease prevalence but rather that it increased medical avoidance for serious conditions unrelated to COVID-19 –13. The While the decline in acute ED visits has been well-documented, there are still questions about which populations experienced significant declines, and by extension, may be at risk for subsequent adverse outcomes. Some empirical evidence suggests that the overall decline in ED utilization was concentrated among certain race/ethnic groups who already face high medical needs. In the early days of the pandemic, there was a large reduction in non-COVID-19 admissions for ZIP codes which were majority-Hispanic, majority-Black, or high poverty , 6. HoweIt is additionally unclear how long this downturn in ED utilization persisted. Understanding the duration of reduced ED care has important health implications, as potential health effects due to the drop in ED visits for acute conditions could be mitigated if visits rebounded after the initial downturn , 24, 25,We used electronic health records (EHR) extracted for ED visits from January 5, 2018, to December 31, 2020, from the Los Angeles County + USC Medical center (LAC + USC), a 600-bed safety-net hospital and the largest safety-net hospital of Los Angeles County. The EHR data were extracted from the LAC + USC Medical Center’s Cerner PowerInsight clinical reporting tool. The patient diagnoses codes were inputted for administrative purposes, including patient care and billing, and were assigned by trained coders at the medical center based on physician documentation. All project activities were reviewed and approved by the University of Southern California Institutional Review Board (HS-19-00890), which served as a reliance for the University of California, Irvine Institutional Review Board.Cardiac emergencies, diabetic complications, and strokes were identified using the Clinical Classifications Software (CCS) for the International Classification of Diseases, Tenth Revision (ICD-10) diagnosis codes. Cardiac emergencies were defined as acute myocardial infarctions (CCS code 100), coronary atherosclerosis and other heart disease (CCS code 101), cardiac dysrhythmias (CCS code 106), and cardiac arrest and ventricular fibrillation (CCS code 107). Patients with diabetic complications were defined as having diabetes mellitus with complications (CCS code 50) including ketoacidosis, hypoglycemia, hyperglycemia, skin ulcers, macular edema, or chronic kidney disease. Stroke emergencies were defined as acute cerebrovascular disease (CCS code 109), occlusion or stenosis of precerebral arteries (CCS code 110) and transient cerebral ischemia (CCS code 112). These emergency conditions were selected because they require timely healthcare to prevent additional health complications and death. These three conditions have also been previously examined using national syndromic surveillance program data to determine the level of ED utilization during the pandemic for specific acute conditions [Our analyses included the 156 weeks between January 5, 2018, and December 31, 2020. We designated the early pandemic period as the eight weeks between March 13, 2020, and May 7, 2020, which started when the Trump Administration issued the national emergency order and ended when California started relaxing social distancing restrictions and businesses were reopening . We desiTo test whether the early and later pandemic periods represented anomalous ED utilization trends than would be expected, we conducted an interrupted time series analysis using autoregressive integrated moving average (ARIMA) . Our maiFor the three medical conditions of interest there were a total of 4864 ED visits between March 13 and July 2, 2020: 2,596 were for cardiac emergencies (53.4%), 2,014 were for diabetic complications (41.4%), and 254 were for strokes (5.2%). During the early pandemic period, total ED visits were 48% lower than in the same period in 2019  and a larger proportion of males visited the ED   and 4.1% for Black patients , while accounting for autocorrelation. The coefficient for Black patients was not significantly different from zero, meaning that the average number of ED visits for acute conditions during the post lock-down was not different from previous weeks.Our study examined the reduction in ED visits at a Los Angeles safety-net hospital during the early COVID-19 pandemic for Black and Hispanic patients. Consistent with other work , 15, we We do not interpret this acute drop in ED visits in Spring 2020 as a reduction in disease incidence because these medical emergencies often result from uncontrolled chronic conditions . The redThis ED avoidance is especially problematic for Black and Hispanic patients, as they have a high prevalence of the underlying conditions –34 that Black patients experienced a rapid rebound in ED visits for acute conditions between May and July 2020, but Hispanic patients continued to see lower-than-expected ED use after the stay-at-home orders were lifted. In California, lockdown restrictions were lifted in early May 2020 , which mAnother possible explanation for the difference in ED rebound magnitude between Black and Hispanic patients is help seeking behaviors, namely the use of telemedicine. The empirical evidence is mixed but some studies found Black patients utilized telemedicine more than non-Black patients during the early pandemic , 36, whiOur paper addresses important questions about the health implications of the widely studied decline in ED use immediately following the COVID-19 national emergency declaration in March 2020 by better characterizing the nature of this slowdown by race/ethnicity and the stability of this trend after stay-at-home orders were lifted. These findings can help researchers and practitioners understand potential health impacts of the well-documented drop in ED visits by identifying most affected patient groups, further characterizing gaps in healthcare utilization, and the magnitude of the rebound in ED use to compensate for the downturn. Importantly, we also improve upon earlier work by using time-series methods to control for well-characterized pre-COVID-19 patterns in ED utilization.There are a few limitations to our study. As we do not have data outside of the ED setting, we do not know whether patients who avoided the ED sought help elsewhere, or if they died with EMS or at home. Moreover, our study did not examine mortality data and therefore it is unclear if there was an increase in deaths due to our medical conditions of interest. Although lack of mortality data was a limitation, documenting the delay in care for these conditions is a strength of the study since delayed treatment can lead to additional complications which adversely influence patients’ lives and burden the healthcare system. These study findings may not generalize to all regions because the population analyzed were patients visiting an ED at a safety net hospital in an urban location. However, these results may be applicable to other safety net hospitals located in regions with a high social vulnerability index. Our study also used a composite variable for the timeseries analysis due to the low individual counts, yet despite this limitation the time-series analysis was an important strength of the study, and we were able to adjust for seasonal trends in ED visits using multi-year data. Further, this study fills the significant gap in research by investigating ED utilization for urgent medical conditions among historically marginalized and low-income groups.Among Hispanic and Black patient, ED visits for cardiac emergencies, diabetic complications, and strokes, declined significantly during the early COVID-19 pandemic period. After societal restrictions were loosened in May 2020, however, there was a gradual rebound among Black patients. The magnitude of the rebound was less pronounced in Hispanic patients. Further exploration into health behaviors is needed to determine what factors motivated Hispanics to continue to avoid medical assistance for time-sensitive, life-threatening conditions, as well as any protective factors that motivated Black patients to seek care after the initial decline. To date, new information is being discovered about the COVID-19 disease and much is still unknown. It is possible that highly contagious COVID-19 subvariants can cause a surge in cases and deaths, which overwhelm EDs and compel governments to reinforce lockdowns similar to March 2020. Health agencies should communicate the importance of seeking medical assistance at EDs for cardiac emergencies, diabetic complications, and strokes, even during lockdowns to minimize treatable conditions and avoidable deaths. Addressing these disparities and encouraging patients to seek medical care for urgent conditions can mitigate the burden on the healthcare system.