We aimed to conduct a narrative synthesis of components and indicators of community vulnerability to a pandemic and discuss their interrelationships from an ecological perspective.We searched from PubMed, Embase, Web of Science, PsycINFO, and Scopus (updated to November 2021) for studies focusing on community vulnerability to a pandemic caused by novel respiratory viruses on a geographic unit basis . Studies that reported the associations of community vulnerability levels with at least one disease morbidity or mortality outcome were included.Forty-one studies were included. All were about the COVID-19 pandemic. Suitable temperature and humidity environments, advanced social and human development , and settings that intensified physical interactions are important indicators of vulnerability to viral exposure. However, the eventual pandemic health impacts are predominant in communities that faced environmental pollution, higher proportions of socioeconomically deprived people, health deprivation, higher proportions of poor-condition households, limited access to preventive health care and urban infrastructure, uneven social and human development, and racism. More stringent social distancing policies were associated with lower COVID-19 morbidity and mortality only in the early pandemic phases. Prolonged social distancing policies can disproportionately burden the socially disadvantaged and racially/ethnically marginalized groups.Community vulnerability to a pandemic is foremost the vulnerability of the ecological systems shaped by complex interactions between the human and environmental systems.PROSPERO (CRD42021266186). COVID-19 afflicted populations globally. Some communities have been disproportionally impacted . A pandeConsiderable research has emerged focusing on measuring community vulnerability to the COVID-19 pandemic -12. MostEvolving insights underline the importance of system-oriented analysis of vulnerability . CommuniWe conducted a narrative literatures synthesis in response to the growing studies on community vulnerability relevant to a pandemic. A narrative synthesis approach was used due to the high heterogeneity in metrics for quantifying community vulnerability, geographic units of community and health outcomes used to validate the vulnerability metrics across relevant studies. A narrative synthesis is more suitable for synthesizing more diverse evidence, allowing for concept mapping to advance theoretical development . Based oThis study was pre-registered with Prospective Register of Systematic Reviews (Reference No.: CRD42021266186) and prepared following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISM) [We targeted studies focusing on community vulnerability in the context of a pandemic or emerging infectious diseases (EIDs) caused by novel respiratory viruses and qualifying as pandemic. Literature was retrieved from five databases  to encompass multiple disciplines. The literature search was performed in December 2021 to capture all articles published up to November 30, 2021, using a combination of search terms related to community vulnerability and emerging infectious diseases (EID) that are caused by novel respiratory viruses. We limited the review to EIDs caused by novel respiratory viruses because these diseases share similar (if not common) transmission modes, control measures, and clinical consequences, thus sharing similar indicators of vulnerability and operations of the systems to shape community vulnerability. Due to the high heterogeneity of terms relating to community vulnerability, we performed a backward/forward literature search, title, abstract, and full-text screening to cover a list of search terms related to community vulnerability as exhaustively as possible. A full list of search terms was shown in Table S1 in the After deduplication, two researchers independently screened the titles and abstracts of retrieved articles based on the inclusion and exclusion criteria  were consistently associated with higher COVID-19 morbidity or mortality. The associations of income and age with COVID-19 morbidity and mortality were more heterogeneous across studies. Poorer health conditions, including greater prevalence of chronic diseases ,28,37,38For household composition, only single-parent household was positively associated with both COVID-19 morbidity and mortality . RegardiHealthcare resources, including primary health care and public health resources ,46,47,49More advanced social and human development, indicated by lower economic inequity, greater life expectancy, higher gross domestic product, and higher literacy rates were associated with higher COVID-19 morbidity ,36,46,49Viral exposure facilitators  ,47-49,52Our study has some limitations. First, as a narrative synthesis, our study may be criticized for its insufficient rigorousness and transparency for quantitative evidence synthesis when compared to a meta-synthesis. However, a narrative synthesis is more suitable for answering broader research questions, synthesizing mixed  evidence, and enabling theoretical development. Second, this review excluded articles that used one single indicator to evaluate vulnerability to a pandemic and articles that did not measure any disease-related morbidity or mortality outcomes, and thus cannot exhaustively identify all potential vulnerability indicators. Third, our article search was restricted to those studies containing key terms loosely relating to “community vulnerability” and may be insufficient to capture a high heterogeneity of environmental contexts that shape community vulnerability to a pandemic. Furthermore, most of the included studies were conducted in the USA or Brazil, which limited the generalizability of the research findings. Increasing the availability and accessibility of relevant data from global to national levels and to smaller geographic unit (such as neighbourhoods) can help stakeholders of different levels to evaluate and manage community vulnerability to future pandemics. Our study may also be subject to publication bias due to restricting to only English published articles.Community vulnerability to a pandemic is the vulnerability of the ecological systems shaped by the complex interactions between the human and environment systems. Environmental factors of suitable temperature and humidity and human factors indicative of more advanced social and human development co-determine community vulnerability to viral exposure. However, the impact of the pandemic was determined by the systems’ biophysical and social resources. Pandemic recovery should focus on building more sustainable and resilient systems rather than simply resuming prior economic activities.


Despite an extensive workup, nearly one third of ischemic strokes are defined as Embolic Stroke of Undetermined Source (ESUS), indicating that no clear etiologic cause has been identified. Since large vessel atherosclerotic disease is a major cause of ischemic stroke, we focus on imaging of large vessel atherosclerosis to identify further sources of potential emboli which may be contributing to ESUS. For a stroke to be considered ESUS, both the extracranial and intracranial vessels must have <50% stenosis. Given the recent paradigm shift in our understanding of the role of plaque vulnerability in ischemic stroke risk, we evaluate the role of imaging specific high-risk extracranial plaque features in non-stenosing plaque and their potential contributions to ESUS. Further, intracranial vessel-wall MR is another potential tool to identify non-stenosing atherosclerotic plaques which may also contribute to ESUS. In this review, we discuss the role of cross-sectional imaging of the extracranial and intracranial arteries and how imaging may potentially uncover high risk plaque features which may be contributing to ischemic strokes. Despite an extensive workup, nearly one-third of ischemic strokes are defined as Embolic Stroke of Undetermined Source (ESUS) meaning that no definite cause of the stroke has been identified . ESUS haCardiac sources, specifically atrial fibrillation, were thought to play a major role in ESUS because occult atrial fibrillation was found in many patients with ESUS . HoweverIn this review article, we will review the role of cross-sectional imaging of the carotid arteries in patients presenting with ESUS. First, we will discuss the current standard of care and typical imaging workup to exclude carotid disease as a potential cause of stroke. We will then discuss the role of computed tomography angiography (CTA) and magnetic resonance angiography (MRA) in evaluating potential causes of ischemic stroke in the extracranial carotid artery. We will also review the role of intracranial vessel wall MR (VW-MR) in assessing intracranial atherosclerosis, another potential contributor to ESUS.Rather than being a diagnosis of exclusion, ESUS has a standardized, criteria-based definition requiring specific imaging and clinical workup. In order to meet criteria for a diagnosis of ESUS, an ischemic stroke must be a non-lacunar stroke detected on CT or MR imaging, the patient must have ≤ 50% luminal stenosis of the extracranial and intracranial vessels supplying the territory of the brain infarction, and have no major risk of a cardioembolic source or other specific identifiable cause of stroke, such as arteritis, dissection, migraine/vasospasm, or drug misuse. In order to make this diagnosis, suggested diagnostic assessment in evaluating those with ESUS is a brain CT or MR, 12-lead electrocardiogram, precordial echocardiography, cardiac monitoring for 24 h with automated rhythm detection, and imaging of both the extracranial and intracranial arteries supplying the area of brain ischemia with either digital subtraction angiography, MR or CT angiography, or cervical duplex and transcranial Doppler ultrasonography , 4.via artery-to-artery embolism  where the degree of stenosis based on flow measurements can be assessed. US can also evaluate various plaque features which are known to be higher risk, including echolucent plaque . Though When imaging the extracranial carotid artery in the setting of ischemic stroke, there are two major considerations. First, the degree of luminal stenosis must be assessed using a standardized system, most commonly using North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria . This caMR is the most studied method to visualize vulnerable plaque features with considerable evidence supporting its use to evaluate advanced atherosclerotic plaque. Intraplaque hemorrhage (IPH) is the most commonly encountered MR-detected plaque feature considered to be “high-risk” and is strongly associated with infarction –17. OtheMultiple studies have specifically evaluated the role of MR imaging of plaque in the setting of ESUS. Several studies have evaluated individuals with ESUS and have found that these MR-detected vulnerable plaque features are more commonly seen ipsilateral to the side of stroke compared to the contralateral side –25. ReceThough MR imaging of the extracranial carotid arteries can be helpful in identifying high risk plaque features, its widespread use is somewhat limited by availability, patient contraindications , and usually lengthy sequences making it a time-consuming imaging examination.CTA is an increasingly commonly used imaging modality in the assessment of etiology of acute ischemic stroke. Because it is relatively cost-effective and quick to obtain, it is most often the first-line examination for those presenting emergently with acute stroke symptoms. While there is more prospective evidence that MR-assessed vulnerable plaque features contribute to future and recurrent ischemic stroke, many of these plaque features can also be assessed using CTA imaging . While MThough not frequently used in everyday practice, there may be a role for more advanced imaging to evaluate for higher risk plaque. Positron Emission Tomography (PET) imaging has been studied as a method for assessing the vulnerability of carotid plaque. A recent systematic review and meta-analysis found that carotid arteries ipsilateral to recent ischemic events had more avid uptake of markers of inflammatory activity  than asymptomatic arteries . Other tIntracranial atherosclerosis leads to up to 9–15% of ischemic infarctions in the United States and up to 50% worldwide. Similar to extracranial atherosclerosis, intracranial atherosclerosis must result in at least 50% narrowing in order to be considered causative in the setting of ischemic stroke. Active atherosclerotic plaque can easily be overlooked when using conventional angiographic imaging because plaques do not always produce associated vessel narrowing. Because of this, intracranial vessel wall MR (VW-MR) can be used as an imaging assessment of atherosclerosis, particularly non-stenosing plaque.Intracranial VW-MR is a powerful tool to image beyond the vessel lumen and for evaluating non-stenosing plaque which may lead to ischemic stroke. In order to accurately assess the vessel wall, there are several critical components to intracranial VW-MR imaging . First, Intracranial VW-MR can more easily detect smaller plaques or plaques with associated positive remodeling which may not produce narrowing on angiographic imaging but may still lead to ischemic stroke. Positive remodeling is an adaptive process where the outer wall of a vessel can outwardly bulge in the setting of an atherosclerotic plaque to preserve cerebral blood flow, leading to a normal, non-stenotic appearance on standard angiographic imaging techniques, including CTA, MRA, and DSA. Positive remodeling is commonly seen in the posterior circulation but can be seen in any intracranial arteries. Because of the common occurrence of positive remodeling, many patients presenting with acute ischemic stroke may have a normal appearing angiographic study without any suspicious findings for a contributing atherosclerotic lesion. When imaged using VW-MR, however, culprit atherosclerotic plaques may be identified  and are VW-MR uses a few specific imaging findings to identify active or culprit atherosclerotic plaques. The most important imaging finding for evaluating plaque in the setting of acute ischemic stroke is plaque enhancement. Several meta-analyses show that plaque/vessel wall enhancement is very strongly associated with culprit or symptomatic plaques –40. PlaqWhen used in patients with ESUS, some studies have found that intracranial VW-MR can be a helpful tool. A study with over 240 patients with ESUS found that intracranial plaque was much more common ipsilateral to the side of stroke . They alIntracranial VW-MR has become increasingly popular in evaluating ischemic stroke and ESUS patients, with a recent survey suggesting that more than 50% of neuroradiology practices routinely perform this type of study . DespiteGiven recent randomized clinical trial findings that treating cardiac sources for ESUS may not be as beneficial as originally hoped, more attention is being placed on other potential embolic sources. Since the current ESUS definitions require <50% luminal narrowing, potential culprit plaques could be missed or inadequately treated because they are producing insignificant narrowing. In the extracranial carotid artery, both MR and CTA can be used to identify certain plaque features which indicate more plaque vulnerability including IPH on MR and increased soft plaque thickness on CTA. VW-MR can also be used as a powerful tool to identify non-stenosing but active atherosclerotic plaque in the intracranial arteries by identifying an enhancing plaque with positive remodeling. Though these studies can be helpful in determining the source of potential emboli, there are some Further studies are needed to validate these imaging techniques and pave a path for their routine use in ESUS.HB, HK, and AG contributed to conception of the manuscript. HB wrote the first draft of the manuscript. HK and AG critically revised and wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.HB is in part supported by National Institutes of Health grant 5U24NS107156-04. AG is in part supported by National Institutes of Health grants R01HL144541 and R01NS123576. HK serves as a PI for the NIH-funded ARCADIA trial (NINDS U01NS095869).® and ancillary study support from Roche Diagnostics; as Deputy Editor for JAMA Neurology; on clinical trial steering/executive committees for Medtronic, Janssen, and Javelin Medical; and on endpoint adjudication committees for AstraZeneca, Novo Nordisk, and Boehringer Ingelheim. He has an ownership interest in TETMedical, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.HK serves as a PI for the NIH-funded ARCADIA trial (NINDS U01NS095869), which receives in-kind study drug from the BMS-Pfizer Alliance for EliquisAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Urogenital tuberculosis (UGTB) can affect the entire urinary tract including the kidneys, ureters (strictures), urinary bladder, prostate in addition to involving reproductive tracts. In modern day practice, both ultrasound and cross-sectional imaging play an important role in the radiological diagnosis of UGTB. The sequalae of untreated UGTB is morbid and can lead to end-stage renal failure, infertility, and life-threatening systemic infection. UGTB is less commonly observed in developed countries and may mimic other pathologies including malignancy. Thus, it is important that radiologists consider the differential diagnosis early, particularly individuals with risk factors such as travel to endemic regions, to allow optimal treatment and ensure best prognostic outcomes. UGTB can typically be managed by Infectious Disease clinicians with multidrug chemotherapy.Mycobacterium tuberculosis infection and thus microbiological diagnosis should be sought to confirm the diagnosis.We have presented a case of microbiologically proven extrapulmonary tuberculosis (TB) predominantly involving the genitourinary tract. The response to TB agents and lack of evidence of co-infection with another organism, might suggest this as the first published case of emphysematous tuberculous prostatitis. Emphysematous prostatitis is indicative of a gas-forming infection of the prostate, and is associated with abscess formation in the vast majority of case and is an easily identified radiological feature on CT. It is not a well-recognised feature of  Oxfordshire has a sizeable East Timorese population with multiple health and social needs. Individuals born in East Timor form the largest group of new TB notifications in Oxfordshire [unpublished data].Tuberculosis (TB) is a multisystemic disease and remains the leading infectious cause of death worldwide. East Timor has a very high incidence of TB, with 508 cases/100,000 population reported in 2020 and the country has is an associated high mortality from TB. but affects just 1.5% of patients with tuberculosis overall. Urogenital tuberculosis (UGTB) is defined as infectious inflammation of urogenital system organs in any combination, caused by Mycobacterium tuberculosis complex. Dissemination from the primary focus of disease occurs via haematogenous, lymphatic or local spread from the principal focus of disease. Testicular tuberculosis is rare and occurs by spread from epididymoorchitis, while seminal vesicles are affected via retrograde canalicular transmission from the prostate or urinary tract. In females, the fallopian tubes, uterine endometrium and ovaries are affected. Signs and symptoms are non-specific including amenorrhea, adnexal masses and lymphadenopathy. Adhesions within the uterus (Asherman’s Syndrome) can lead to menstrual dysfunction and infertility. Case reports indicate that urogenital TB may be sexually transmitted.Lymph node TB (LNTB) is the most frequent form of extrapulmonary TB. The genitourinary tract is another common site of extrapulmonary tuberculosis in the developing world (where over 90% of cases are seen) The disease is frequently considered an imitator of other diseases such as urinary tract infection and malignancy, leading to incorrect or delayed diagnosis and subsequent complications such as infertility and renal failure. Therefore, non-specific urogenital presentations or features suggestive of malignancy amongst high-risk patients should prompt clinicians to maintain a high index of suspicion for UGTB.While the definite diagnosis of UGTB remains widely based on early morning urine cultures and histopathology, diagnosis is challenging and delayed, especially in low prevalence countries including the United Kingdom. However, it can deceptively involve the prostate in immunocompetent individuals, mimicking cancer, emphasising the importance of including UGTB as a differential in high-risk patients. Tuberculous prostatitis is rare and the complication of prostatic abscess is extremely rare.Tuberculous prostatitis has mainly been associated with the immunocompromised patient. In the context of immunosuppression, UGTB may manifest as sepsis, with bacteraemia and visceral metastatic foci. There is a case report of a patient presenting with emphysematous cystitis with pre-existing pulmonary TB, although the association is unclear and immunosuppressive status and diabetes may have been contributing factors. To date, there are no other published cases of emphysematous prostatitis due to TB.Untreated UGTB has been previously shown to lead to ureteral strictures, contracted bladder, obstructive nephropathy, renal parenchymal infiltration and ultimately end stage renal failure. In complex cases a multidisciplinary approach involving urology and/or radiological procedures may be required, e.g. for tissue diagnosis, debridement of necrotic tissue or abscess drainage.UGTB is usually managed by Infectious Disease clinicians with multidrug chemotherapy, infrequent use of surgery and strict follow-up. Recent literature highlights the key role of imaging as a method to assess the extent of disease involvement, identifying complications and guidance for interventional requirement such as biopsies or drainage.The value of cross-sectional imaging towards the evaluation of UGTB has been widely described and most recently proved pivotal towards raising suspicion of tuberculosis, although it has been highlighted that radiological appearances of UGTB are typically indistinguishable from those of other infectious causes. Within the testes and epididymis, while some lesions demonstrate hyper or mixed echotexture, most of these lesions are hypoechoic. Occasionally, hypoechoic nodules are seen within the enlarged epididymis, displaying a miliary pattern. Progression of disease into the scrotal skin and tunica vaginalis may lead to hydrocele, pyocele, scrotal wall thickening and sinus formation. Tuberculous prostatitis is rare in the immunocompetent and may present with prostate gland enlargement with poorly defined hypoechoic lesions, usually in the posterolateral peripheral zones and irregularity of the external contour of the prostate. Colour doppler flow is increased in both tuberculous epidydimitis and prostatitis.High resolution ultrasound has been described as the best imaging modality for assessing the epididymis, testis, scrotum and vas deferens to detect pathology such as inflammation, abscesses, large granulomas and calcification. Chronic findings of untreated UGTB may include contracted urinary bladder.Cross-sectional imaging (CT or MRI) has a significant role in identification of abscesses, lymphadenopathy, pulmonary involvement, musculoskeletal complications and soft tissue or parenchymal abnormalities in suspected cases of disseminated TB. Hydronephrosis can be severe when the renal pelvis and ureter are affected. Radiological CT features have included renal caliectasis, papillary necrosis and healing after the acute stage may include multifocal strictures. In established tuberculosis, the kidney atrophies and loses its structure, appearing as thin-walled cysts or as a multiloculated cyst.T2 weighted images with regards to solid caseation, fibrosis and calcification on MRI. T2 hyperintensity has been observed with oedema or abscess formation. Prostate tuberculosis may appear as nodular and diffuse. In the former, the peripheral zone shows one or multiple T2-hypointense areas, whereas the diffuse pattern is typified by diffuse glandular enlargement. Abscesses are demonstrated as T2-hyperintense foci with central diffusion restriction and rim-enhancement.Comparable to TB elsewhere, most cases demonstrate hypointense signal on A 30-year-old male, originally from East Timor who moved to UK 6 years previously, attended the Emergency Department with lower abdominal pain and dysuria. Urinalysis at that time demonstrated leucocytes and nitrites and he was treated for a lower urinary tract infection. 4 weeks later, he reattended the Emergency Department with left iliac fossa pain and dysuria for 3 days on a background of 4 weeks of increasing painful testicular swelling. Following discussion with the Urology team, the patient was referred for a testicular ultrasound to exclude testicular pathology including epididymitis. Ultrasound demonstrated a heterogenous lesion which appeared to be centred on the left epididymis but extending into the left testis. At this time, the differential diagnoses included infection (epididymo-orchitis) with a left testicular abscess, and the possibility of TB was raised, and a paratesticular tumour was deemed less likely. Tumour markers including alpha-feta protein (AFP) and human chorionic gonadotrophin (hCG) and circulating testosterone levels were all within normal limits.The patient was discussed at the germ cell multidisciplinary team meeting where there was broad agreement with the initial differential diagnosis. TB was again considered as a possible causative agent. The patient was commenced on a 2-week course of (broad-spectrum) ciprofloxacin and doxycycline for an atypical orchitis, a regime which has potential for some anti-TB activity. Subsequent serial ultrasound scans demonstrated new lesions in the right testis and progression of the left testicular changes.Subsequently, a contrast-enhanced CT was performed, with several salient imaging findings, which provided further evidence to support a radiological diagnosis of TB. The patient was referred on to the Infectious Diseases (ID) department for ongoing management. Microbiological confirmation was obtained by TB polymerase chain reaction (PCR) and culture of pus expressed from a scrotal sinus.9/L). The eosinophilia was found to be the result of coinfection with Strongyloides, treated successfully with Ivermectin.The patient had very mildly raised inflammatory markers throughout the clinical course with a C-Reactive Protein (CRP) of 21.6 (at presentation) and 17.8 mg/L (at the time of diagnosis). His full blood count (FBC) was normal other than a persistent, mild, eosinophilia  with a significant reduction in his main symptom, scrotal swelling. Following a period where the patient returned to East Timor and was lost to earlier follow-up, the patient had a follow-up CT scan 8 months following the initial CT scan, with complete radiological resolution of the GU tract findings. The patient did not receive further treatment agents during this time.Serial testicular ultrasound scans revealed progressive epididymis and testicular hypoechoic lesions, in keeping with TB abscesses, initially confined to the left epididymis, subsequently progressing to left testis and then bilaterally with scrotal thickening . Whilst The CT abdomen and pelvis demonstrates hypoperfusion in the upper pole of the left kidney without hydronephrosis, consistent with direct infection of the renal parenchyma with TB. The remainder of the renal parenchyma was normally enhancing .Correlating with the ultrasound findings , there iMycobacterium tuberculosis (Mtb) complex with no molecular markers for rifampicin resistance. Pus was cultured in Mycobacterial Growth Indicator Tubes and positive growth was confirmed as Mtb by lateral flow. Whole genome sequencing at the National Mycobacterium Reference Service (South) confirmed the presence of Mtb and susceptibility to first-line anti-tuberculosis therapy and quinolones. There was no evidence for co-infection with another organism, although deeper (prostatic) sampling was not performed.Microbiological confirmation was obtained from pus expressed from a sinus on the scrotum. Rapid diagnosis was obtained by Cepheid MTB/RIF TB PCR: this confirmed the presence of Mtb infection and thus microbiological diagnosis should be sought to confirm the diagnosis. The response to TB agents and lack of evidence of co-infection with another organism might suggest this as the first published case of emphysematous prostatitis due to TB.We have presented a case of microbiologically proven extrapulmonary TB predominantly involving the genitourinary tract, including emphysematous tuberculous prostatitis, which is an uncommon clinical finding and may be neglected as a radiological diagnosis. Emphysematous prostatitis is an easily identified radiological feature on CT imaging. It is indicative of a gas-forming infection of the prostate, and which is associated with abscess formation in the vast majority of cases. It is not be a well-recognised feature of UGTB remains rare in developed countriesUGTB an often presents with non-specific symptoms and can mimic other pathologies such as renal tract malignancyEarly radiological recognition of UGTB is key to early diagnosis and improved outcomesIt is important to have a high-index of suspicion for UGTB in patients from endemic regionsUGTB is typically managed by Infectious Disease clinicians with multidrug chemotherapyA multidisciplinary approach involving urology and/or radiological procedures may be required in complex casesStrict follow-up following treatment is crucial given the often indolent nature of systemic TBTuberculous prostatitis is rare and the complication of prostatic abscess is extremely rareWe believe this may be the first reported case of emphysematous tuberculous prostatitis.


In spite of astonishing advances in epilepsy research and treatment over the past few decades, epilepsy remains one of the most common and devastating brain diseases and still affects approximately 65 million people globally . In addide novo status epilepticus (SE), brain infections, traumatic brain injuries, brain tumors, and strokes . Recent studies have uncovered a variety of mechanisms whereby oxidative stress and neuroinflammation greatly influence each other in the context of epilepsy. For instance, neuroinflammation can be regulated by transcription factors such as NF-κB and nrf2 that are activated by reactive oxygen species (ROS). Neuroinflammation in turn can induce the expression and activity of NADPH oxidase (NOX), fostering a highly oxidative environment. Moreover, the oxidative and proinflammatory mediators can moulate distinct intracellular pathways expressed in different cell-types, exemplified by NOX-2 dependent increase in ROS in neurons and astrocytes triggered by SE, and myeloid differentiation primary response 88 (MyD88) dependent glial activation through Toll-like receptors (TLRs) . The reviews presented in this collection highlight how signaling crosstalk between neuroinflammation and oxidative stress and their cell type specific roles may be leveraged for novel therapeutic strategies for epilepsy.Both inflammation and oxidative stress are well known for their pathophysiological roles in the epileptic brain. However, they are often studied as separate entities despite the evidence that the redox-based signaling cascades and inflammatory reactions have extensive crosstalk  infection to generate acute brain inflammation and the subsequent spontaneous seizures in mice. They show TMEV infection-induced seizures likely due to impaired GABAergic inhibition, secondary to alterations in neuronal intracellular chloride regulation. Their results further suggest that the brain-derived neurotrophic factor (BDNF) might contribute to the development of brain infection-triggered seizures by reducing the expression of K+/Cl− cotransporter 2 (KCC2). This has the potential to enhance accumulation of intracellular chloride and increases excitability by rendering GABA depolarizing instead of hyperpolarizing as observed in chemoconvulsant models of SE , a structural analog of type G chemical warfare agents , is well known to induce SE, gliosis, neuronal death, and eventually the development of spontaneous recurrent seizures in rodents. Jia et al. demonstrate that the conventional ASD, valproate, combined with low-intensity exercise can reduce seizures and associated co-morbidities in kainate-treated mice. The reduction in seizure burden appears to be correlated with the suppression of inflammatory cytokines  and the immune receptor TLR4 in the hippocampus. Given that TLR4 is involved in epileptogenesis of diverse etiologies (The benefits of low-intensity physical exercise to the CNS have been shown in animal models and patients with neurological diseases, such as Alzheimer’s disease, Parkinson’s disease, stroke, epilepsy, multiple sclerosis, anxiety and depression . Jia et iologies , these fSakamoto et al. propose a diagnostic algorithm that might help to predict the underlying autoimmune etiology of epilepsy before antibody testing results become available. Over 30% of epilepsy patients suffer from pharmacoresistant seizures associated with cognitive and psychiatric co-morbidities. Analyzing a microarray dataset from the Gene Expression Omnibus database, Min et al. identify 25 genes differentially expressed in the peripheral blood of patients with valproate resistance in epilepsy and significantly enriched in T-cell receptor recognition. While the potential confound posed by the differential seizure burden between valproate sensitive and resistant groups needs to be considered, these findings suggest that the peripheral blood T-cells and the differentially expressed genes could serve as biomarkers for refractory epilepsy. Identification of reliable biomarkers for diverse types of epilepsy and pharmacoresistance could facilitate both early diagnosis and development of new therapies, needed to achieve the ultimate goals of “no seizures, no side effects, and no co-morbidities” in epilepsy treatment.Developing preventive treatment for epilepsy is challenging because it is currently impossible to identify individuals that will develop epilepsy after initial precipitating brain insults. Theoretically, biomarkers that identify “at risk” individuals would facilitate the development of potential antiepileptogenic treatment . By reviThe series of articles presented here address diverse ways in which neuroinflammation could shape acquired epilepsy and offers insights into how these processes may be leveraged to inform mechanisms of epileptogenesis, identify biomarkers, and to develop novel strategies for disease modification and treatment in epilepsy.


Dysregulated microRNA (miRNA) expression could provide a mechanism linking firefighter exposure to increased cancer risk.To determine if changes in longitudinal miRNA expression in firefighters are associated with occupational exposures.Whole blood MiRNA was evaluated in 52 new recruits prior to live-fire training and 20–37 months later. Linear mixed effects models adjusted for age, ethnicity, BMI, and batch effects were used to determine associations separately for all fires and structure fires only between employment duration, cumulative fire-hours and fire-runs, and time since most recent fire with (1) nine a priori and (2) the full array of 799 miRNAs.For multivariable models including all fires, two a priori miRNAs were associated with employment duration and four with time since most recent fire. For multivariable models restricted to structure fires, three a priori miRNAs were associated with employment duration and one with fire-runs. Additional miRNAs from the full array were associated with employment duration for all fires and/or structure fires. In general, tumor suppressive miRNAs decreased and oncogenic miRNAs increased with exposure.Changes in miRNAs may serve as biomarkers of exposure effects and a mechanism for increased cancer risk in firefighters. Firefighting is a hazardous occupation involving exposure to toxic combustion byproducts, including many known and probable carcinogens –5. WithiDespite the number of epidemiologic studies linking firefighting to excess cancer risk, there is limited information on the cellular mechanisms that lead to cancer in firefighters. Partly due to this lack of mechanistic evidence, an assessment by the International Agency for Research on Cancer concluded that firefighting was only possibly carcinogenic in humans . EpigeneMiRNAs are small non-coding RNAs involved in the regulation of cell cycle progression, apoptosis, and cell differentiation, with reported oncogenic or tumor-suppressive roles, though these may vary depending on which gene is targeted and which cancer is considered –21. Our In our current analysis we evaluated whole blood samples, collected from newly recruited firefighters prior to live-fire training and again ~2 years later, for miRNA expression levels and determined if changes in expression were associated with measures of occupational exposure. We hypothesized that after ~2 years of employment in the fire service, we would be able to identify differential miRNA expression patterns among newly recruited firefighters that were associated with employment duration, surrogate measures of fire exposure (cumulative hours at fire and cumulative number of fires), and/or most recent fire exposure, and that changes in these miRNAs would be associated with increased cancer risk based on published studies in the general population.2) and classified using World Health Organization categories: normal (18.5–24.9), overweight (25.0–29.9) and obese (≥30). Biological samples including blood, urine and buccal cells were collected at baseline and blood samples were collected again after a period of 20 to 37 months, at which time a follow-up survey was administered. Employment duration, the total months between baseline and follow-up, was calculated.All study protocols and materials were approved by the University of Arizona Institutional Review Board (IRB). Study participants provided written informed consent. New-recruit firefighters with no previous live-fire exposures were recruited from the Tucson Fire Department (TFD) between 2015 and 2016. Participant age, race, ethnicity, height, and weight were collected from health records and current tobacco usage was assessed at study enrollment via survey. Participants who reported current tobacco usage were excluded from analyses. Height and weight were used to calculated body mass index (BMI) (kg/mCumulative hours at fire (fire-hours) and cumulative number of fires (fire-runs) were collected from TFD response records and assessed as surrogate measures of chronic fireground exposure. This information included the type of fire , date and time of the fire, and duration of the fire response (minutes). The time between the blood draw at follow-up and the new recruit’s most recent response to a fire call (days since most recent fire exposure) was calculated. Fire-hours, fire-runs, and days since most recent fire exposure were determined for: (1) all fire types ; (2) structure fires only; and (3) non-structure fires only. Other fires, or non-structure non-vehicle fires, consisted of outside vegetation  fires, outside trash fires, outside fires involving property of value , and unclassified fires.The protocols used for sample collection and processing have been previously reported . Levels ruv” R package (https://cran.r-project.org/package=ruv). RUV-III corrected miRNAs were then analyzed using the limma software package  pyrene  pyrene , one of ] pyrene . Based o] pyrene  may contSurrogate fireground measures have been associated with cancer risk among firefighters in a pooled-cohort analysis of three metropolitan career fire departments in the US, including significantly increased lung cancer and mortality risk associated with fire-hours and similar but non-significant associations with fire-runs as well as a marginally significant association between leukemia mortality and fire-runs but not fire-hours . In our We identified nine novel miRNAs with at least a 25% fold longitudinal change associated with employment duration in our full array analyses restricted to structural fires. Four of the five miRNAs with decreased expression are reported as being tumor suppressors and the four with increased expression are reported as being oncogenic in various cancers, including colorectal, liver, prostate, breast, and bone cancers , 39–41. In most of our multivariable analyses, we did not observe significant associations between surrogate measures of fireground exposure and dysregulated miRNA expression. Given the relatively small sample size of our current study, we likely lacked sufficient power to detect additional significant associations. However, the majority of firefighter responses are to medical aid calls , and othThe current study findings are consistent with both chronic and acute contributions of firefighter exposures to longitudinal change in whole blood miRNA. Relative to our prior study comparing whole blood miRNA in incumbent firefighters with an average of 14.1 years of occupational exposure compared to new recruits , the folTo our knowledge, this is the first longitudinal analysis of epigenetic changes in firefighters and the first longitudinal assessment of surrogate fireground exposures associated with changes in miRNA expression. We were able to enroll a cohort of new-recruit firefighters with no previous occupational smoke exposure and collect baseline blood samples prior to live-fire training, allowing us to capture a more accurate assessment of miRNA expression prior to any fireground exposures for comparison to samples collected ~2 years later. Collection of fire response history for each study participant allowed us to examine potential dose-response relationships between surrogate fireground exposures and change in miRNA expression as well as evaluate and adjust chronic surrogate measures of fireground exposure (cumulative fire-hours and fire-runs) for potential confounding by acute fireground exposures (days between follow-up blood draw and most recent fire response).Despite our study’s strengths, there were limitations in regards to both exposure assessment and miRNA measurement. Though we were able to capture administrative information regarding type of fire and our study sample was limited to firefighters within the first two or three years of their career , we were unable to account for potential confounding by job assignment . In regards to miRNA measurement, our samples collected at baseline and follow-up were analyzed in four separate batches. To address batch effects, we employed the newer RUV-III correction approach, rather than older methods of batch effect correction such as the ComBat function . Use of In conclusion, consistent with previous research, our study provides further evidence that miRNAs may serve as biomarkers of cumulative exposure to firefighters, although the influence of acute exposures and the entirety of firefighter occupational exposures  requires further investigation. Additionally, we observed significant dose-response relationships between employment duration and dysregulated expression of a priori miRNAs implicated in cancers and cancer pathways. Together, our study provides evidence that alterations of miRNA expression may serve as a mechanism linking firefighter exposures to increased cancer risk.Supplementary Information for: Longitudinal evaluation of whole blood miRNA expression in firefighters


Stroke is one of the leading causes of death and disability worldwide . The appEmerging reperfusion therapies in acute ischemic stroke (AIS) as well as evolving strategies to reverse anticoagulation in hemorrhagic stroke require early differentiation of stroke type. Frequently used emergency imaging such as computed tomography (CT) distinguishes ischemic and hemorrhagic stroke and thereby opens the avenue for recanalization therapies if intracranial hematoma is absent. Nonetheless, such imaging techniques are typically unavailable in the prehospital setting; CT-scans are also not sensitive to show ischemic brain lesions in the hyperacute setting. Experimental investigations proposed microRNA (miRNA) and exosomes as well as metabolites as markers of cerebral ischemia that might support AIS diagnosis. However, translational data in humans are sparse.Prognostication after stroke depends on the interplay between demographic factors , stroke subtype, and stroke etiology, as well as clinical severity. Persisting disability following ischemic stroke is a result of neuronal death, network dysfunction, and synaptic loss; specific markers of neuronal damage showed superiority over others in predicting functional outcome after stroke , 3, wherOverall, there is a clear need for experimental as well as translational research on blood-based molecular and cellular biomarkers for differentiation of stroke and stroke mimics, stroke types (AIS vs. hemorrhagic stroke), to guide individual treatment decisions and provide information to patients and relatives. The same applies for the determination of stroke etiology and for the better understanding of secondary neuronal damage in order to develop new treatment paradigms within the hyperacute phase of AIS and hemorrhagic stroke.microRNAs are small non-coding RNAs with a length of ~22 nucleotides that regulate gene expression by destabilizing and repression translation of complementary mRNAs. They are characterized by a high expression in mammalian brains and are involved in modulation of excitotoxicity, microglia polarization, oxidative stress, neuronal apoptosis, and oxidative stress, all together mechanisms orchestrating secondary brain damage and thereby regulating functional recovery following AIS , 6. TherFrom a diagnostic point of view, an increasing number of studies have identified stroke-specific patterns of circulating miRNAs that were also associated with symptom severity as well as infarct volume and predicted functional outcome. Patients with AIS showed higher circulating levels of miR-125a-5p, miR-125b-5p, and miRNA-143-3p compared to patients with transient ischemic attack (TIA) or neurologically normal subjects with a return to control levels 2 days after symptom onset, supporting the utility of these miRNAs for early stroke diagnosis in the emergency setting . FurtherCepparulo et al. compared miRNA levels between animal models of ischemic stroke  and hemorrhagic stroke (collagenase-induced hemorrhagic stroke). They demonstrated upregulation of specific miRNAs as early as 3 h after the procedure in these distinct animal models, pointing toward a potential clinical usage in differentiating ischemic from hemorrhagic stroke in the clinical setting.In a translational approach, Besides miRNA, “Omics” reflecting specific pathophysiologic aspects of ischemic as well as hemorrhagic stroke have been investigated as potential biomarkers for stroke diagnosis, differentiation of ischemic vs. hemorrhagic stroke, prediction of functional outcome, and risk of stroke recurrence.Malicek et al. aimed to unravel potential new candidates for differentiation of ischemic and hemorrhagic stroke by using an exploratory proteomic-based pilot study. They identified nine potential candidates connected with the immune system, the coagulation cascade and apoptotic processes. These markers now have to be validated in larger cohorts during the hyperacute phase and with additional analysis such as ELISA, Western Blot, and Mass spectrometry. These efforts aim to shorten treatment delay in stroke patients eligible for recanalization therapies by enabling prehospital differentiation of stroke subtypes.Within this Topic Section collection of articles, Kirzinger et al.; Li et al.; Sun et al.), markers for oxidative stress , vasoactive peptides , and BBB function  are investigated. In addition to these markers reflecting specific pathophysiologic aspects of ischemic/hemorrhagic stroke, individual patient outcome is additionally determined by stroke-associated complications such as infections  and cardiac comorbidities . Beside their usefulness in predicting stroke prognosis, markers of cardiac pathology might also be useful in uncovering stroke etiology, as it is reported for natriuretic peptides and their role in identifying patients with atrial fibrillation  as a marker for endothelial dysfunction, to be associated with stroke recurrence and progression of cerebral white matter lesions.Patients suffering from stroke experience a higher rate of stroke recurrence. Identifying patients with increased risk of stroke recurrence might help to improve secondary preventive strategies and to select patients for intensified stroke etiology work-up and prevention support programs. Exosomes 30–150 nm) belong to the family of extracellular vesicles, together with shedding microvesicles  and apopototic bodies . Emerging evidence underlines a potential role of exosomes as diagnostic, therapeutic, and prognostic marker in stroke. Exosomes are endosome-derived vesicles; the following steps describe their formation: initiation, endocytosis, multivesicular body formation, and exosome secretion. Exosome secretion means the final process, in which the previously formed multivesicular bodies are fused with the plasma membrane and are finally secreted by their cell of origin. As the intracellular origin of secreted exosomes is seldom demonstrated, the term “small extracellular vesicle” may be more accurate , but “ex0 nm beloDue to the fact that exosomes are abundantly secreted by most human cells, they play an exceptional role in intercellular signaling via cell-to-cell communication. Moreover, they are characterized by a lipid bilayer with an aqueous core, thereby harboring the ability to cross the BBB and transport various molecules across the BBB . This feWith this in mind, exosome-derived markers might be a powerful tool for distinction of stroke subtypes and obtaining information on the pathophysiologic state within the CNS. So far, multiple exosome-derived miRNAs were associated with ischemic stroke outcome [e.g., miRNA-223 , miRNA-1Jödicke et al. analyzed dynamic changes of extracellular vesicles in the biomarker and perfusion-training-induced changes after stroke (BAPTISe) study and uncovered an association with functional outcome in patients with subacute stroke.Within the current article collection, The current Research Topic, “Blood-Based Biomarkers in Acute Ischemic Stroke and Hemorrhagic Stroke”  providesTU, HA, MG, ST, and RK edited the Research Topic and drafted and revised the editorial manuscript. All authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intrinsically disordered proteins (IDPs) are highly prevalent and play important roles in biology and human diseases. It is now also recognized that many IDPs remain dynamic even in specific complexes and functional assemblies. Computer simulations are essential for deriving a molecular description of the disordered protein ensembles and dynamic interactions for a mechanistic understanding of IDPs in biology, diseases, and therapeutics. Here, we provide an in-depth review of recent advances in the multi-scale simulation of disordered protein states, with a particular emphasis on the development and application of advanced sampling techniques for studying IDPs. These techniques are critical for adequate sampling of the manifold functionally relevant conformational spaces of IDPs. Together with dramatically improved protein force fields, these advanced simulation approaches have achieved substantial success and demonstrated significant promise towards the quantitative and predictive modeling of IDPs and their dynamic interactions. We will also discuss important challenges remaining in the atomistic simulation of larger systems and how various coarse-grained approaches may help to bridge the remaining gaps in the accessible time- and length-scales of IDP simulations. Intrinsically disordered proteins (IDPs) or regions (IDRs), compared to well-structured proteins, do not have stable tertiary structures under physiological conditions. Nevertheless, IDPs or IDRs can be found in nearly a third of proteins encoded in the human proteome , and theIt is very challenging to provide reliable descriptions of the conformational ensembles of IDPs and IDRs. A disordered state does not lend itself to traditional structural determination methods that are geared toward describing a coherent set of similar structures. Biophysical techniques, such as NMR, SAXS, and FRET, can provide complementary information on various local and long-range structural organizations . Howeverde novo simulations of disordered protein ensembles require both high force field accuracy and adequate sampling of relevant conformational space, pushing the limit of these two central ingredients of molecular dynamics (MD) and Monte Carlo (MC) simulations. The challenges of simulating disordered proteins have driven significant interest in developing better protein force fields and advanced sampling methods , and then use the Metropolis rule to allow MC to exchange the replicas and maintain the detailed balance. A key advantage of using multiple replicas and maintaining detailed balance is avoiding the reweighting problem generally required for biased simulations. Note that virtually all biased sampling strategies can be readily incorporated within the REX framework to benefit from both classes of enhanced sampling, including metadynamics (MTD) [Enhanced sampling techniques generally accelerate the crossing of energy barriers to achieve better sampling efficiency, such as by introducing bias potentials, modifying the potential energy itself, and changing the effective temperature. These techniques have proven essential in atomistic simulations of IDPs ,71, yielcs (MTD) ,73, accecs (MTD) , umbrellcs (MTD) ,76, and cs (MTD) . In praccs (MTD) . Here, wMTD and its variants have been considered one of the most important collective variables (CV)-based sampling methods for protein simulations . MTD useA central limitation of CV-based sampling methods is that the efficiency strongly depends on the quality of selected CV(s). For diffusion processes such as protein conformational fluctuation, it is often not clear which CVs can best capture large-scale transitions or even if these transitions could be effectively described using one or a few CVs ,96,97. ACV-free sampling avoids the need to identify a set of optimal CVs and can be highly desirable for simulating high-dimensional conformational fluctuation of IDPs. Many CV-free sampling methods have also been developed, including the tempering-based and energy-scaled biased methods. Tempering-based sampling methods rely on increasing the effective simulation temperature  to accelerate barrier crossing. Examples include the temperature cool walking , annealeaMD adds boost potentials to reduce the energy barriers and accelerate sampling . HoweverX is the conformational coordinates and βm is the inverse of kBTm. The scaling of ‘solute’-‘solute’ interactions allows the ‘solute’ to be simulated with an effective temperature of Tm while maintaining the ‘solvent’ temperature at T0. As a result, the exchange acceptance probability will be independent of ‘solvent’–‘solvent’ interactions, which reduces the effective system size and requires fewer replicas to cover the same temperature range. A key open choice in REST is how the ‘solute’–‘solvent’ term is scaled (Equation (1)). Different solute–solute and solute–solvent scaling factors can strongly affect the ability of driving conformational transitions of the selected ‘solute’ region. A strong solute–solute interaction favors to compact the protein conformations, whereas a strong solute–solvent interaction prefers the disordered, solvent-exposed conformations. Different scaling schemes lead to very different characteristics of REST1  and REST2 (revised) protocols (Equation (1)). High temperature conditions favor the unfolded conformations in REST1, while both folded and unfolded conformations were observed in REST2 model for the condition with the same effective ‘solute’ temperature. The reason for this is that REST2 was designed to have a weaker solute-solvent interactions to promote the sampling of folded conformations even at high temperatures [REST is a special variant of T-REMD designed specifically to reduce the number of DOFs that contribute to the Metropolis criteria of replica exchange, such that smaller number of replicas is needed ,85. The eratures . Separateratures . REST has proven to be one of the most reliable choices for enhanced sampling of protein folding and particularly disordered conformational ensembles ,114. SugDespite the success of REST for CV-free enhanced sampling, it does not benefit from targeted acceleration along specific CVs that are known to be rate limiting. For this, REST  has been combined with CV-based enhanced sampling to maximize the efficiency of sampling the complex, high dimensional conformational space of proteins. Some of the examples are discussed in the sections above. Here, we note a couple additional recent examples. By integrating free energy perturbation (FEP) and REST methods, Abel et al. obtained more thorough samplings of different ligand conformations around the active site and realized relative binding affinity predictions . OkamotoMultiscale enhanced sampling (MSES) is yet another fascinating example of a CV-free enhanced sampling strategy. Protein folding and other cooperative transitions such as self-assembly are known to be dominated by entropy barriers, which renders tempering ineffective for driving faster transitions. Coupled with a lack of obvious CVs, sampling complex conformational transitions of IDPs and their interactions is challenging for both CV-based and REX-based CV-free methods. For this, an effective solution is to couple atomistic simulations with a coarse-grain (CG) model, such that one could benefit from both faster transitions of CG modeling and accuracy of atomistic force field . A partiOther tempering methods including integrated tempering and simulated tempering have also been combined with different biased potentials to enhance sampling ,128. ForkBT), especially for the near-Gaussian biased unitless potentials [When bias potentials are used to enhance sampling, reweighting is often required to obtain the unbiased samples and construct statistically optimal unbiased free energy surfaces. Two reweighting methods are widely used for this, including the weighted histogram analysis method (WHAM) for the biased simulations with specific CVs and a more general multistate Bennett acceptance ratio (MBAR) approach ,130. Thetentials . This aptentials . It shoutentials ).As discussed above, dramatic improvement in atomistic protein force fields coupled with enhanced samplings and GPU computing have now enabled us to generate the disordered conformational ensembles of increasingly complex IDPs in both bound and unbound states. Many important phenomena related to IDPs remain largely out of the reach of physics-based atomistic simulations, such as aggregation ,133,134 Implicit treatment of solvent is an effective approach to reduce the computational cost of atomistic IDP simulations. The basic idea is to directly estimate the solvation free energy to capture the mean effect of solvent on the thermodynamic properties of the solute . ImpliciDespite many successes, implicit solvent models have not widely been tested and applied to the studies of larger IDPs. Several factors likely contribute to this. Most implicit solvent models are built upon existing protein force fields, which until recent years have had significant limitations in describing disordered protein conformations. Implicit treatment of solvent also relies on various approximations for computational efficiency, such as treating water as a continuous dielectric medium in GB models, limiting the ability of implicit solvent to accurately capture the conformational dependence of solvation free energy. A particular limitation is the common use of a surface area (SA)-based model to describe nonpolar solvation energy, which has known limitations in describing the length-scale dependence as well as solvent screening of dispersion interactions . These lSeveral recent efforts have been made to further improve implicit solvent models for IDP simulations. The GB-Neck2 model has been optimized to reproduce solvation energies for a variety of protein systems . Recent Notwithstanding the ever-improving atomistic modeling, coarse-graining has remained an attractive and often effective strategy for extending the accessible time and length-scales of MD simulations. By grouping multiple (protein) atoms into CG beads and using simplified potential energy functions, CG modeling does not only reduce the system size, often by ~10-fold, but also allows much larger MD integration time steps up to 10 s of fs. Together, many CG models can be several orders of magnitude more efficient than atomistic ones. Numerous CG models have achieved varying levels of success in studies of protein folding, binding, and assembly ,163. NonGō/Gō-like models, also known as topology-based models, are based on the funneled energy landscape theory  and have⍺, Cβ, and O atoms, and has been reparametrized for IDPs by adjusting the secondary structure-related potential energy terms as well as introducing a new parameter, VRg term, for controlling the collapse and size fluctuation of the protein. Several higher resolution coarse-grained models have been also developed specifically for modeling IDPs. Thirumalai and co-workers reparametrize the two-bead self-organized polymer coarse-grained model (SOP-CG) to reproduce Rg values of a set of diverse IDPs with 20 to 441 residues . The res⍺-only CG model to represent the disordered low complexity domain of the RNA-binding protein FUS-LCD and the DEAD-box helicase protein LAF-1 in the formation of LLPS [⍺-only CG models in molecular simulations of LLPS involving IDPs. Nonetheless, difficulty in describing local structure propensities (such as transient helices) with the C⍺-only representation may be an important limitation for studying certain specific effects of IDPs in LLPS. An important application for CG models is to study liquid-liquid phase transitions (LLPS) that are frequently mediated by IDPs ,44,45,46 of LLPS . The mod of LLPS  or the K of LLPS  for shor of LLPS , which a of LLPS . The res of LLPS . The resEffective and reliable molecular simulations are crucial for characterizing the details of disordered conformational ensembles of IDPs in isolation, dynamic complexes, or biological condensates. Such computational capability, integrated with experimental studies, makes it possible to determine how the dynamic protein states may respond various cellular stimuli in signaling and regulation and more rigorously establish the (dynamic) structure-function relationship of IDPs and IDRs. In this review, we highlight recent advances in meeting two central requirements for reliable IDP simulations, namely accurate force fields, for describing the energetics of protein conformations, and efficient MD simulation methods, for the adequate sampling of relevant conformational space. The need to simulate disordered protein ensembles has played a key role in driving significant improvements in empirical protein force fields in recent years. Many of these force fields are now well balanced for both folded and disordered proteins. The force field development itself has directly benefited from many advanced sampling methods that allow for accurate calculation of the conformational equilibria of model peptides and proteins during force field recalibration. These enhanced sampling techniques rely on carefully designed biasing potentials, modification to the original Hamiltonian, and/or tempering to accelerate barrier crossing and generate statistically meaningful ensembles with far less computation. Many of the enhanced sampling strategies are complementary and can be readily integrated together to further improve the efficiency. Together, the improved protein force fields and powerful sampling techniques now allow realistic simulations of the conformation and interaction of at least modest-sized IDPs at the atomistic level. Nonetheless, the high dimensionality and complex nature of disordered protein conformation continues to push the limits of the force field and sampling capability. In particular, none of these methods alone appears to be generally applicable to simulate IDPs that are large  and/or contain nontrivial residual structural features. There remains an urgent need and exciting opportunities in developing much more effective methods for sampling IDP conformations and dynamic interactions, such as through the careful integration of various existing CV-dependent and CV-free strategies. A particular promising direction is to leverage machine learning to design superior adaptive sampling strategies that can generate optimal bias potentials on the fly to maximally drive the exploration of the free energy landscape. ⍺-only single-bead protein models to implicit solvent ones with atomistic proteins. Many of the current models are geared towards modeling systems with minimal residual structures. A key challenge in the multi-scale modeling and simulation of IDPs is finding the optimal compromise between resolution, accuracy, and efficiency for the particular problem of interest. Nonetheless, it can be expected that multi-scale simulations will continue to play a central role in studying IDPs and dynamic interactions. Many proteins models with various levels of resolution are also being developed and fine-tuned for IDP simulations, particularly for studying biological condensates. These models range from C


Among low- and middle-income countries (LMICs), bipolar disorder is recognized as one of the leading causes of disease burden for adults and is associated with marked suicide risk. There are limited data on suicidal ideation in bipolar disorder from LMICs. This study presents cross-sectional data on the prevalence of suicidality and associated patient characteristics among patients with bipolar depression in Pakistan, a lower-middle income country and the fifth most populous country in the world.Participants were recruited through outpatient psychiatric clinics in between 2016–2019 in Karachi, Lahore, Hyderabad and Rawalpindi between 2016–2019. Participants were aged 18 to 65 years with a known diagnosis of bipolar disorder and currently in a depressive episode. Suicidality was assessed using the suicide item of the 17-item Hamilton Depression Rating Scale (HAM-D) and levels of severity were categorized as absent, mild/moderate, or severe. Biometric data and biomarkers were obtained. Descriptive statistics were used to describe prevalence and proportional odds regression models were applied to establish correlates to suicidal ideation.Among the 266 participants, 67% indicated suicidality of any level and 16% endorsed severe suicidality. Lower body mass index (BMI) , higher HAM-D score , lower C-reactive protein (CRP) level , and increased number of inpatient hospitalizations  were identified as significant predictors of suicidality in the fully adjusted regression model. No patient demographic data, including age, gender, marital status, socioeconomic status, and years of education were associated with severity of suicidality.There exists a high prevalence of suicidal ideation among patients with bipolar depression in Pakistan. Our findings add to the limited literature on suicidality in bipolar disorder in the LMIC context and suggest roles of biological variables such as BMI and CRP level in predicting suicidal ideation and potentially suicidal behaviours in bipolar depression. More studies are needed to see whether such findings can be replicated in other similar LMIC settings, and to explore potential physiological pathways linking BMI, inflammatory biomarkers and suicidality in bipolar disorder.


Bone morphogenetic proteins (BMPs), which compose the largest group of the transforming growth factor-β (TGF-ß) superfamily, have been implied to play a crucial role in diverse physiological processes. The most intriguing feature of BMP signaling is that it elicits heterogeneous responses from cells with equivalent identity, thus permitting highly context-dependent signaling outcomes. In endothelial cells (ECs), which are increasingly perceived as a highly heterogeneous population of cells with respect to their morphology, function, as well as molecular characteristics, BMP signaling has shown to elicit diverse and often opposite effects, illustrating the innate complexity of signaling responses. In this review, we provide a concise yet comprehensive overview of how outcomes of BMP signaling are modulated in a context-dependent manner with an emphasis on the underlying molecular mechanisms and summarize how these regulations of the BMP signaling promote endothelial heterogeneity. Endothelial cells (ECs) exhibit great heterogeneity , in theiAs one of the archetypal receptor-ligand-mediated signaling pathways, bone morphogenetic protein (BMP) signaling has gained increasing importance in the development and homeostasis of the vascular system. Its ability to selectively activate ECs in a context-dependent manner without influencing the survival of ECs  satisfieTo better understand how BMP signaling modulates endothelial heterogeneity, it is essential to understand how BMP signaling functions within ECs. However, a simplified schematic diagram fails to capture the complex molecular landscape of BMP signaling, since distinct expression patterns of individual BMP signaling components result in diverse context-dependent outcomes . In addiAlk3 within naïve ECs led to the failure of venous specific marker expression in presumptive venous ECs  and lymphatic ECs (LECs), the two major subtypes of ECs . TherefoEven within BECs, it has been shown that the responsiveness toward BMP signaling also varies between arterial and venous ECs. For instance, we and others have shown that pro-angiogenic BMP signaling could selectively induce angiogenic responses in venous ECs without affecting arterial ECs . TogetheBone morphogenetic protein signaling seems to be highly context-dependent, eliciting opposite outcomes in similar cell types . It appein vivo, computational analyses revealed that cells distinguish distinct signaling environments, such as the concentration of ligands and differences in ligand–receptor complexes, to respond to BMP stimulation. Interestingly, inside the signaling-receiving cells, the canonical downstream target SMAD1/5/8 is activated regardless of the signaling-conferring ligand–receptor complex, suggesting the possibility that signaling-receiving cells are only capable of perceiving the sum of BMP ligand stimulation  of SMAD1 . In addiof SMAD1 . Moreoveof SMAD1 . Furtherof SMAD1 . Recent of SMAD1 . TherefoPost-translational modifications (PTMs) have been shown to contribute to the extensive versatility and complexity of signaling pathways in eukaryotic cells. Perturbations of PTMs have been linked to various diseases, illustrating the significance of PTMs in maintaining cellular homeostasis . While PIn addition to phosphorylation, ubiquitination and sumoylation, both of which are reversible PTM, have been shown to modulate the context of BMP signaling  Figure . The priO-linked N-acetyl-glucosamine (O-GlcNAc) moiety to target proteins, has been shown to fine-tune Drosophila BMP type I receptor Saxophone (Sax), an ortholog for human ALK2 , which contributes to the development of vascular lesions (Numerous studies have suggested that dysregulation of BMP signaling in ECs could lead to various diseases. For instance, mutations in BMPR2 predispose to the onset of PAH , a rare CCMs . DeletioCCMs . Moreove lesions , hinting lesions . In addi lesions . TherefoAs we obtain comprehensive knowledge on the role of BMP signaling in endothelial heterogeneity, we are confronted with a plethora of enigmas. Increasing evidence indicates that the complexity of BMP signaling dynamics provides essential regulation on endothelial plasticity. Emerging reports have identified novel fine-tuning regulatory mechanisms for BMP signaling. While their functions remain largely unknown in the endothelium, given the ubiquitous expression of these regulators, it is plausible that BMP signaling in ECs is similarly modulated. Since the role of BMP signaling in endothelial heterogeneity is still in its infancy, it will be of great importance to elucidate distinct regulatory mechanisms and more importantly its impact on the endothelial phenotype, which ultimately will lead to distinct physiological responses in the body. The plethora of regulatory possibilities provide an additional dimension for regulating distinct BMP signaling components and therefore further enrich the impact of the BMP signaling on endothelial heterogeneity. Not yet identified environmental factors, as well as various PTMs, might give us answers to yet unsolved enigmas and offer novel strategies to overcome limitations faced so far. Therefore, further investigations will provide insights into how such a conceptually simple signaling cascade can give rise to exquisite plasticity and versatility. Moreover, the availability of novel techniques, such as NGS, single-cell analysis, and bioinformatics, will refine our understanding of endothelial dysfunctions and concomitant diseases and ultimately contribute to the development of new therapeutic interventions for various diseases by fully harnessing the heterogeneity and context-dependent properties of BMP signaling.All authors wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myocardial infarctions are one of the most common forms of cardiac injury and death worldwide. Infarctions cause immediate necrosis in a localized region of the myocardium, which is followed by a repair process with inflammatory, proliferative, and maturation phases. This repair process culminates in the formation of scar tissue, which often leads to heart failure in the months or years after the initial injury. In each reparative phase, the infarct microenvironment is characterized by distinct biochemical, physical, and mechanical features, such as inflammatory cytokine production, localized hypoxia, and tissue stiffening, which likely each contribute to physiological and pathological tissue remodeling by mechanisms that are incompletely understood. Traditionally, simplified two-dimensional cell culture systems or animal models have been implemented to elucidate basic pathophysiological mechanisms or predict drug responses following myocardial infarction. However, these conventional approaches offer limited spatiotemporal control over relevant features of the post-infarct cellular microenvironment. To address these gaps, Organ on a Chip models of post-infarct myocardium have recently emerged as new paradigms for dissecting the highly complex, heterogeneous, and dynamic post-infarct microenvironment. In this review, we describe recent Organ on a Chip models of post-infarct myocardium, including their limitations and future opportunities in disease modeling and drug screening. Cardiovascular disease is the leading cause of death worldwide, responsible for over 17.9 million deaths annually –4.When a patient presents with a myocardial infarction, percutaneous coronary intervention is often performed, which is a catheterization procedure to restore flow to the interrupted artery and minimize the initial insult , 6. RapiOne challenge facing the development of new therapies for post-infarct myocardium is that conventional preclinical models are limited primarily to non-human animal models or static, uniform monolayers of cultured cells that poorly replicate the clinical setting, especially the complex, dynamic remodeling that occurs post-infarction . Thus, din vitro systems that mimic the fundamental structural and functional units of native tissues and have been shown to mimic responses to drugs at clinical doses in various tissue models with higher fidelity than conventional in vitro methods  and 3-dimensional (3-D) tissue constructs. Lastly, we will describe considerations for future work to promote clinical mimicry and translation of Organ on a Chip models of myocardial infarction.The initial coronary occlusion of a myocardial infarction deprives downstream myocardium of vital oxygen and nutrients, causing cardiac myocyte necrosis within hours , 12, 13.As a result of infarct expansion, diastolic and systolic wall stresses increase , 18. To Tissue necrosis and infarct expansion can also increase the likelihood of myocardial rupture , 15. BecThe proliferative phase occurs over the next few weeks and is characterized by cardiac fibroblast proliferation, migration into the site of injury, and differentiation into an activated myofibroblast phenotype. Cardiac fibroblasts are the most abundant non-myocyte cell population in the mammalian myocardium  and, aftThe final phase is scar maturation, which occurs on a timescale of weeks to months. In this phase, myofibroblasts initiate collagen turnover and begin secreting type I collagen, which has the tensile strength of steel , in placIn summary, post-infarct myocardium is characterized by multiple biochemical and biomechanical properties changing in both space and time, which are correlated to complex remodeling of several cell types and the extracellular matrix in parallel . Some orin vitro systems that can modulate oxygen concentrations in space or time have more recently been developed to mimic the hypoxic landscape of post-infarct myocardium. In this section, we will describe conventional hypoxia models that replicate uniform hypoxia as well as engineered systems that offer spatial or temporal control over oxygen tension.Due to the high metabolic activity of cardiac myocytes, the myocardium is a highly vascularized tissue, with capillaries separated by approximately 20 μm . This trin vitro , a family of transcription factors that facilitates the cellular response to hypoxia by upregulating genes associated with survival in low oxygen. In normoxia, HIF is constantly degraded through hydroxylation of the HIF-α subunit by the enzyme prolyl hydroxylase, which marks it for ubiquitination by Von Hippel Lindau protein and subsequent degradation. Cobalt chloride chemically stabilizes HIF in normoxia by replacing Feactivity . Hypoxiaw oxygen , 32, mayw oxygen , and likin vitro models have shown that hypoxia results in structural and functional changes that may contribute to the development of arrhythmias. Specifically, the gap junction protein connexin 43 (Cx43) is known to be affected, which plays a critical role in conducting electrical impulses in the myocardium. Hypoxia has been shown to drive electrical uncoupling in vitro, with effects including decreased Cx43 signal at gap junctions  , 52, insin vitro systems have been engineered. In one example, a microfluidic device was fabricated with a central channel designated for cell culture embedded between two lateral media channels. By flowing media containing a chemical hypoxia mimetic through one channel and standard media through the other, a chemical hypoxia gradient was established in the central cell-containing channel. Cardiac myoblasts near the hypoxic end of the gradient exhibited altered morphology, including reduced cell area and actin disintegration, which was accompanied by mitochondrial dysfunction and decreased cell viability  and was reduced in the absence of cardiac myocytes or with the pharmacological inhibition of TGF-β . Other mLastly, cardiac spheroids have also been implemented to mimic hypoxia gradients. Cardiac spheroids are 3-D aggregates of cardiac cells that recapitulate select aspects of native tissue structure and function –76. BecaIn vitro models have also been engineered to replicate dynamic changes in oxygen characteristic of ischemia-reperfusion. Microfluidic devices are particularly suitable for this application because they contain chambers of small volumes that can be rapidly filled with hypoxic gas or cell medium. For example, a microfluidic device with integrated bioelectronics was used to measure intracellular action potential and extracellular beat rate and propagation velocity in cardiac myocytes cultured in a microchannel. The microchannel was rapidly filled with hypoxic cell medium followed by recovery medium to mimic ischemia-reperfusion. Hypoxic cardiac myocytes demonstrated substantially reduced depolarization times and beat rates, as well as irregular propagation patterns, which recovered within 30 min after reintroducing normoxic cell media , which is biocompatible, mechanically tunable, and transparent . A. Ain sitin vivo. To more closely mimic cell-cell and cell-matrix interactions that occur in native myocardium, cardiac cells have been mixed into hydrogels to form 3-D tissues. Similar to findings in 2-D, cardiac myocytes encapsulated in rigid polyethylene glycol hydrogels demonstrate reduced cell shortening and increased relaxation time when compared with soft hydrogels, which was also accompanied by increased intracellular localization of the mechanosensitive transcription factor YAP  or pathophysiologic (>20 kPa) tissues. Cardiac myocytes in stiff, fibrous networks exhibit slower calcium flux, indicated by increased decay time and increased peak-to-peak irregularity  or fibrotic (61.1 kPa) myocardium, cardiac myocytes were cocultured with 25 or 75% cardiac fibroblasts, respectively. Fibrotic tissues underwent more rapid compaction and had higher collagen content, disrupted myofibril structures, altered Cx43 distribution, prolonged time to peak, and lower contractile force generation when compared with healthy tissues. To next create a spatially heterogeneous stiffness model, which can mimic the interface between the infarct zone and viable tissue, fibrotic and healthy tissue were integrated at opposing sides of a single biowire platform . The fibTo alter substrate rigidity over time, one approach is to encapsulate cells into hydrogels that either degrade or cross-link in response to specific wavelengths of light. In one example, a 3-D photodegradable hydrogel was used to demonstrate that valvular myofibroblasts can be redirected into a quiescent phenotype by decreasing stiffness. This work demonstrates fibroblast phenotypic plasticity and the potential role of the mechanical environment in de-differentiating fibroblasts, which has therapeutic applications in resolving fibrotic disease . Lastly,in vitro, experimental platforms include microchips with stretchable silicone membranes, custom-built bioreactors, or commercially available cell straining units in which strain can be applied to cell culture plates with integrated loading posts.Cardiac cells are constantly under cyclic stretch in the healthy, beating heart. Myocardial infarction results in an initial loss of contractility in the infarct zone followed by arrhythmogenesis, which alters strain rates experienced by surviving cardiac cells, as quantified through echocardiographic imaging . To streChronic cyclic stretch over several days to mimic the diastolic and systolic movement of cardiac muscle has been shown to promote the maturation of “engineered heart tissues,” which are generally defined as cardiac myocytes embedded in hydrogels and cast around uniaxial tissue constraints or circular molds. Stretched heart tissues exhibit increased cell alignment –151, sarCardiac fibroblast responses to strain have also been relatively inconsistent. In some cases, stretching activates many hallmarks of cardiac fibrosis, including increased fibroblast proliferation, hydrogel stiffening , increasTo assess the combined effects of strain and hypoxia, cardiac fibroblasts have been cultured in a microfluidic device containing a stretchable, gas-permeable membrane situated above a microchannel for gas flow and between lateral actuation channels . Uniformin vivo. In this device, coculture with stretched cardiac myocytes increases cardiac fibroblast proliferation. A media screen indicated the presence of cytokines such as colony stimulating factor 1 and platelet derived growth factor B, which were sufficient to increase proliferation in fibroblast monocultures , which have historically been the most accessible cardiac cell source. Cells from neonatal rats exhibit species-specific differences and are relatively resistant to hypoxia, a key feature of the infarct microenvironment . Thus, mLastly, Organ on a Chip systems need to be more high-throughput and scalable to be integrated into the drug discovery pipeline. Thus, the field also needs more scalable fabrication methods, such as rapid, multimaterial bioprinting of cardiac biowire scaffolds into 96-well plate formats  or the dIn summary, engineered Organ on a Chip models of post-infarct myocardium have exciting potential to address many of the gaps presented by oversimplified 2-D cell culture models and animal models that lack human relevance. As technologies continue to develop, next-generation multi-dimensional models could provide simultaneous control over spatial and temporal changes in the physical, biochemical, and mechanical microenvironment that correspond to the phases of infarct healing. When further advanced with patient-derived cells, scalable fabrication techniques, and integrated sensors, these models have potential to emerge as new standards for disease modeling and drug screening and lead to new breakthrough therapies for mitigating post-infarction remodeling.Both authors were involved in the conceptualization and writing of this manuscript.MM is an inventor on US patent US9857356B2 filed by Harvard University and licensed to Emulate, Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Goal: Create a semi-automated, graphical, stand-alone application that uses clinically available asynchronous pressure and Doppler velocity captures to rapidly calculate, display, and interpret the pulmonary vascular impedance (PVZ) spectra. Methods: MATLAB-based software was written to analyze PVZ by creating a composite PVZ (cPVZ) spectra comprised of asynchronous screen captures of pulmonary arterial pressure and pulmonary arterial pulsed-wave Doppler velocity waveforms obtained during standard of care procedures. The pressure waveform, Doppler frequency envelopes, and ECG signals were re-digitized via automated border detection. cPVZ of averaged representative beats was calculated in the frequency domain via Fast Fourier Transform, and plotted vs harmonic z. Results: Successful generation of impedance spectra (PVZ(z)), where z is the harmonic, and additional parameters for characteristic impedance (Zc) and stiffness (Zs) were calculated as the mean of PVZ(2-4), and the sum of PVZ , respectively. Conclusions: A graphically driven analysis of PVZ, calculated from standard of care right heart catheterization and echocardiography is possible. This system can help characterize both the steady and pulsatile components of right ventricular (RV) afterload in the clinical setting. I.Overload of the right ventricle (RV) frequently leads to heart failure and death. Therefore, characterizing the full afterload encountered by the RV is critical. The pulmonary vascular impedance (PVZ) spectrum, graphed in the frequency domain, is the only way to fully characterize the combined steady and pulsatile afterload faced by the right ventricle (RV), providing novel insights for diagnosis and treatment II.Our MATLAB-based CompositePVZ© software  analyzesrd harmonic As previously reported, our group has used semi-automated waypoint-driven tracing of waveforms to re-digitize retrospective images of waveforms III.IV.A graphically driven analysis of PVZ, calculated from standard of care right heart catheterization and echocardiography is possible. Our graphical user interface is designed to be similar to the catheterization software already found in the catheterization lab. The similarities will enable clinicians to rapidly select captures of pressure, flow, and resulting spectra to assess the afterload faced by a patient's RV. However, due to the asynchronous manner in which the data is obtained, care should be taken to minimize time between TTE or TEE and right heart catheterization and to ensure that heart rates and rhythms are similar to avoid error. Future versions will also facilitate variable Zc harmonics as high-fidelity wires become more accessible. Multiple potential clinical applications at advanced heart failure and pulmonary hypertension centers exist including drug studies V.In conclusion, a rapid, graphically driven system for calculation of PVZ is feasible. A system such as this will allow full characterization of the RV afterload in the clinical setting, providing clinicians with a tool to characterize patients with pulmonary vascular disease and likelihood of RV failure.VI.A flow diagram of the steps required for PVZ analysis using CompositePVZ© software can be found in the supplemental materials section online. Also, a video of the full analysis which resulted in