Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread. Coronaviridae family are enveloped with a positive-sense single-stranded RNA genome and helical nucleocapsid ) for binding to PDZ domain #2 of ZO1, leading to the inability of claudins to organize into actual barrier-forming strands [Several pathogens, including respiratory viruses, may cause breakdown of cellular barriers  in the lung by a mechanism involving an interaction between the virus and lung epithelial cell TJs to induce leak . These p strands , 46. Alt strands . In addi strands , 49. By  strands . The autOngoing studies will determine whether full length SARS-CoV-2 E interacts with endogenous ZO1 in virus infected cells, in an effort to establish the biological significance of this virus-host interaction. In addition, experiments are underway with lentivirus particles engineered to express the SARS-CoV-2 WT and mutant E proteins alone in human lung airway cells  and in intestinal models (CACO-2 cells) to investigate the E/ZO1 interaction in isolation and its potential effect on TJ integrity via TEER analysis and transepithelial diffusion of paracellular probe molecules. Validation of the E/ZO1 interaction in virus infected cells will be important for future development of small molecule compounds to therapeutically lessen lung and/or GI morbidity and disease symptoms in severe COVID19 patients. Targeting the E/ZO1 interface for treatment may effectively reduce barrier cell damage and diminish the morbidity and mortality associated with this major health threat.


Antimicrobial resistance is highly prevalent in low-income and middle-income countries. International travel contributes substantially to the global spread of intestinal multidrug-resistant Gram-negative bacteria. Hundreds of millions of annual visitors to low-income and middle-income countries are all exposed to intestinal multidrug-resistant Gram-negative bacteria resulting in 30–70% of them being colonised at their return. The colonisation process in high-exposure environments is poorly documented because data have only been derived from before travel and after travel sampling. We characterised colonisation dynamics by exploring daily stool samples while visiting a low-income and middle-income countries.In this prospective, daily, real-time sampling study 20 European visitors to Laos volunteered to provide daily stool samples and completed daily questionnaires for 22 days. Samples were initially assessed at Mahosot Hospital, Vientiane, Laos, for acquisition of extended-spectrum β-lactamase-producing (ESBL) Gram-negative bacteria followed by whole-genome sequencing of isolates at MicrobesNG, University of Birmingham, Birmingham, UK. The primary outcome of the study was to obtain data on the dynamics of intestinal multidrug-resistant bacteria acquisition.Escherichia coli, 10 Klebsiella spp, and 20 other ESBL-producing Gram-negative bacteria), some were shared by as many as four (20%) participants.Between Sept 18 and Sept 20, 2015, 23 volunteers were recruited, of whom 20 (87%) European volunteers were included in the final study population. Although colonisation rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-producing Gram-negative bacteria at some point during the study period; the colonisation status varied day by day. Whole-genome sequencing analysis ascribed the transient pattern of colonisation to sequential acquisition of new strains, resulting in a loss of detectable colonisation by the initial multidrug-resistant Gram-negative strains. 19 (95%) participants acquired two to seven strains. Of the 83 unique strains identified (53 To our knowledge, this is the first study to characterise in real-time the dynamics of acquiring multidrug-resistant Gram-negative bacterial colonisation during travel. Our data show multiple transient colonisation events indicative of constant microbial competition and suggest that travellers are exposed to a greater burden of multidrug-resistant bacteria than previously thought. The data emphasise the need for preventing travellers' diarrhoea and limiting antibiotic use, addressing the two major factors predisposing colonisation.The Finnish Governmental Subsidy for Health Science Research, The Scandinavian Society for Antimicrobial Chemotherapy, the Sigrid Jusélius Foundation, Biotechnology and Biological Sciences Research Council; Wellcome Trust, Medical Research Council; The Royal Society; Joint Programming Initiative on Antimicrobial Resistance, and European Research Council. Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae raises an alarming public health concern.E coli sequence type (ST)131, has been the primary driver of the increased prevalence of extended-spectrum β-lactamase (ESBL)-producing E coli in clinical isolates.E coli, such as ST410K pneumoniae clones, such as CG258 and ST11,Antimicrobial resistance poses a serious threat to human health worldwide.E coli bacteria are the most common, with 30–70% of travellers colonised.E coli strains alongside new non-multidrug-resistant strains, so that the pretravel population remains but as a minority.The literature suggests international travel is strongly associated with the acquisition of multidrug-resistant Gram-negative strains; ESBL-producing Evidence before this studyWe searched PubMed using the terms “beta-lactamase [MeSH]”, and “travel” from the inception of the database to Dec 31, 2019, for human studies in any language. Over the past decade, several investigations reported a high incidence of travel-associated colonisation by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae for travellers returning from tropical and subtropical countries. Apart from destination, travellers' diarrhoea and antibiotic use abroad were identified as major factors predisposing to colonisation. So far, studies exploring travel-acquired ESBL- producing Enterobacteriaceae have focused on findings in post-travel stool. Only one of the investigations reviewed covered the initial stages of acquisition, analysing colonisation dynamics over time, but this study did not use real-time analyses or whole-genome sequencing.Added value of this studyTo our knowledge, this is the first report characterising in real-time the dynamics of ESBL-producing Enterobacteriaceae colonisation during travel to low-income and middle-income countries, with daily stool specimens immediately analysed in a modern microbiology laboratory (Wellcome Trust) on location. Sampling solely after travel would have yielded a multidrug-resistant Gram-negative colonisation rate of 70%, but daily specimen collection revealed a 100% colonisation rate. The acquisition was found to be transient in nature, with detectable colonisation generally lasting one or a few days at most. By analysing isolates from selective primary stool cultures by whole-genome sequencing, the number of individual strains contracted was found to be much higher than anticipated. 19 of 20 participants acquired several different ESBL-producing Enterobacteriaceae strains (2–7 per participant), with a total of 83 individual strains. Our data show that the numerous studies of travellers' colonisation rates have, to date, underestimated the burden. When sampling only before and after travel, the actual burden of multidrug-resistant bacteria is much smaller than it really is because many episodes of multidrug-resistant Gram-negative acquisition lead to transient colonisation were no longer verifiable after travel. Revealing this transient pattern of colonisation where sequential acquisitions of new strains far exceed the figures previously reported, our data considerably widen the current understanding of multidrug-resistant acquisition abroad.Implications of all the evidence availableOur novel observation of multiple transient multidrug-resistant acquisitions in visitors to low-income and middle-income countries unravels the dynamics of the colonisation event. Its transient nature implies a competition between resistant strains and an individual's own microbiota, as suggested by the generally rapid disappearance of the bacteria soon after return to a high-income country. Besides revealing the antimicrobial resistance burden abroad, the surprisingly high number of multidrug-resistant strains acquired might indicate a greater risk for travellers than previously thought because each of these strains might have the potential to transfer resistance genes to members of the individuals' microbiota. Mechanisms underlying the competition between clones should be scrutinised in more detail and methods fight off colonisation already at initial stages must be identified.In previous studies of travellers' intestinal colonisation by multidrug-resistant Gram-negative bacteria, samples have been taken immediately before travel and upon return home.To obtain data on the dynamics of intestinal multidrug-resistant bacteria acquisition, we characterised the colonisation process by ESBL-producing Gram-negative bacteria by sampling participants' stools on arrival in Vientiane, Laos, each day abroad, and at departure. The study was done over 22 days.Volunteers were recruited prospectively from participants at a medical course held from Sept 19 to Oct 9, 2015, in Vientiane. We also invited the recruitees' companions to volunteer. Each participant was asked to provide daily stool samples while staying in Vientiane. Those collected within the first 2 days were considered as baseline samples and the final stool sample was considered the departure samples. Questionnaires assessing background information and travel-related data were used at recruitment and before departure. During the stay, the volunteers were asked to complete a health card to record gastrointestinal symptoms, food habits, and medication use each day. Travellers' diarrhoea was defined as passage of three or more loose or liquid stools per day. Any ESBL-producing Gram-negative bacteria not found in the baseline samples but detected in one or more stool samples taken later were defined as travel-acquired ESBL-producing Gram-negative bacteria. Only volunteers providing at least five daily samples over the 22-day period were included in the final cohort. The study protocol  was apprThe initial screening for presumptive ESBL-producing Gram-negative bacteria strains from stool samples was done at the Microbiology Laboratory of Mahosot Hospital, Vientiane, Laos by culture on CHROMagar ESBL agar plates . Phenotypically distinct colonies were subcultured and stored at −80°C in Protect tubes  with the original swabs. The samples were then transported by plane on dry ice first to the Swiss Tropical and Public Health Institute, Basel, Switzerland, and then to the University of Helsinki, Helsinki, Finland, for analyses. In Finland, the bacteria were recultured on an equivalent chromogenic media, ChromID ESBL agar , and a colony of all morphotypes present on the ChromID plate was subcultured and cryopreserved. The isolates were shipped on dry ice to the University of Birmingham, Birmingham, UK, for genome sequencing by the MicrobesNG facility. Libraries were prepared using the Nextera XT kit  and sequenced on the Illumina HiSeq platform (Illumina).Illumina genome sequence reads were assessed for quality using FastQC (version 0.11.9) and subsequently trimmed using Trimmomatic (version 0.3),Antibiotic resistance genes were detected in assembled and annotated genomes using Abricate (version 0.8.7) and the ResFinder database. Abricate detected all genes with a minimum identity of 75% and a minimum coverage of 0%. Partial hits to genes were then manually assessed because of the occasional splitting of genes (particularly on plasmids). Genes that were present in the total assembly at a coverage of 90% were determined to be present. Prokka-annotated genomes were manually inspected to confirm the presence of resistance genes identified. MLST (version 2.15) was used to verify species identification and assign classical ST designations to isolates. Phylogroups were assigned using ClermonTyping.Isolates were examined for potential relationships by criteria including participant number, ST, resistance profile, and phylogenetic distribution. Where isolates were suspected of sharing recent source or transmission events, Snippy (version 4.3.6) was used to map reads of isolates against the assembled genome of the earliest relative isolate. The assemblies of all isolates within identified clusters were highly similar . The numE coli isolates were screened for virulence factors using the ecoli_vf and vfdb databases bundled in the base version of Abricate. These isolates were then assigned to pathotypes using a previously described schemePhylogenies were reconstructed using RaxML-NG under the GTR-GAMMA model (version 0.6.0), a core single nucleotide polymorphism alignment from Snippy-core (version 4.3.6). Phylogenies were midpoint rooted and combined with metadata for visualisation in Phandango (version 1.3.0). The primary outcome of the study is real-time data on the dynamics of intestinal multidrug-resistant bacteria acquisition while visiting low-income and middle-income countriesThe funders had no role in study design, data collection, data analysis, data interpretation, or writing of this Article.Between Sept 18 and Sept 20, 2015, a total of 23 volunteers were recruited , three oDuring the sampling period, the volunteers stayed at three separate hotels, visited various restaurants either in small groups or all together, and participated in daily rounds at local hospitals. Four (20%) participants reported travellers' diarrhoea and one (5%) took antibiotics .E coli was the most abundant species isolated, accounting for 219 (75%) of the 292 isolates. The other species identified were Citrobacter spp (28 [10%] isolates), Klebsiella spp (16 [5%] isolates), Acinetobacter spp (12 [4%] isolates), and Enterobacter cloacae (11 [4%] isolates); a number of other low-prevalence species, including Aeromonas spp and Stenotrophomonas maltophilia were also present (ESBL-producing Gram-negative bacteria strains were detected in the primary samples of seven (35%) participants, including three who had arrived in Laos directly from another Asian country. For all participants at least one stool specimen tested positive for ESBL-producing Gram-negative bacteria in culture by day 10 of their visit . Of the  present . When alblaCTX-Mfound in a total of 226 (77%) isolates, with CTX-M-55 (64 [28%]), CTX-M-14 (58 [26%]), CTX-M-159 (57 [25%]), CTX-M-15 (30 [13%]), and CTX-M-102 (25 [11%]) the most common types (E coli (one Aeromonas sp and one other K pneumoniae). Superimposing MLST (multilocus sequence typing) designation  participants were colonised by multiple E coli STs and co-colonised by other ESBL-producing Gram-negative bacteria species. For example, one participant took azithromycin for travellers' diarrhoea from Sept 21 to 23, 2015, and was transiently colonised by five different E coli strains during the study, each with different bla gene repertoires. Participant 16 had a regular flux between isolation of an ST38, ST93, and ST101 strain, whereas participant 40 had initial colonisation by an ST48 strain, which was later displaced by an ST38 strain. All these strains have unique signatures of carriage of multiple bla genes, suggesting that travellers are exposed to a large number of multidrug-resistant Gram-negative bacteria and multidrug-resistance conferring genes during the initial colonisation process  containing a Virulence associated gene profile and ST designation consistent with multidrug-resistant atypical enteropathogenic E coli strains  participants, with between zero and five single nucleotide polymorphisms between shared strains. Although direct transmission cannot be confirmed, the clonality of the isolates suggests that the two colonisation events did not result from exposure to a common environmental reservoir. Such reservoirs are generally colonised by bacteria for extended periods of time, which leads to extensive diversity within the bacterial population.E coli isolates in this study has an unexpected composition. Epidemiological surveys of multidrug-resistant E coli, especially those focusing on ESBL strains, are dominated by E coli ST131.E coli in Laos have also shown ST131 to be the dominant drug-resistant lineage in the country.E coli population in Laos might be particularly dynamic and prone to frequent fluctuation. Of note, ST101, ST34, and ST195, and other lineages frequently isolated in our study have never been reported as clinical ESBL-producing E coli isolates in other countries, including the UK where high-quality longitudinal data are available. Some reports describe ESBL-producing E coli ST101,E coli ST38 has been extensively reported as an ESBL-producing strain isolated both from humans and animals.E coli strains were extraintestinal pathogenic (enteropathogenic E coli strains), indicating their potential role as pathogens.The population of ESBL-producing E coli. A few studies have reported multiple findings of ESBL-producing diarrhoeagenic E coliKlebsiellaE coli.E coli (219 [75%] of 292 strains) a substantial number of ESBL-producing non-E coli Gram-negative bacteria, such as Citrobacter (9%), Klebsiella (5%), Acinetobacter (4%), and Enterobacter cloacae (4%) and even low numbers of Aeromonas spp and Stenotrophomonas maltophilia were identified. This variety in the acquired strains might be ascribed to the rate of exposure to various multidrug-resistant Gram-negative bacteria because 19 participants attended a course of tropical medicine, which included daily clinical rounds at local hospitals. High multidrug-resistant Gram-negative colonisation rates have been reported in visitors hospitalised in the tropics.Most previous studies of travellers have only described acquisition of ESBL-producing E coli in these samples is also of note. We screened for ESBL-producing strains, but carbapenemase producers would also have grown on the selective plates used. Additionally, whole-genome sequencing analysis showed a complete absence of carbapenemase genes. This finding was somewhat surprising in southeast Asia where the prevalence of carbapenem-resistant Enterobacteriaceae is increasing.The complete absence of carbapenemase-producing E coli isolates. The ST101 lineage—which dominated our isolate collection—has been identified as a driving lineage in the emergence of ESBL-producing E coli and mobile colistin resistance-positive E coli in the south and southeast Asia,E coli linages surveyed to date. This confirms earlier reports showing that travel exacerbates the global spread of ESBL genes and E coli strains carrying mobile colistin resistance genes.Even more striking were the extremely high concentrations of the mobile colistin resistance gene in our Exposure at hospitals can be considered a limitation of the study because exposure to multidrug-resistant strains is more common in this setting. However, the main aim of the study was to explore the process of colonisation in a high-exposure setting. Although hospital visits might have increased the acquisition rates transient acquisition of multiple strains was also seen for two participants not visiting hospitals, and confirmed in our later studies of regular travellers over a 12-day trip to Benin .By combining real-time sampling of travellers with genome-level analyses, we have shown that colonisation by ESBL-producing Gram-negative bacteria in a high-exposure setting is an extremely dynamic process characterised by competition between resistant strains and an individual's own microbiota. The data suggest that some strains can colonise multiple participants potentially through direct transmission or acquisition from a common source. The challenge now lies in unravelling the mechanisms that underlie this process and competition between the clones, and finding tools to prevent colonisation already at its initial stages.Raw sequence data for all isolates is available via National Center for Biotechnology Information under the Bioproject accession number PRJNA558187.


Since the start of the spread of the coronavirus disease 2019 (COVID-19) pandemic, an international effort has sought to better characterize associated extra-pulmonary health sequelae. The acute and or chronic detrimental impact of SARS-CoV-2 infection on mental health, especially depression, is increasingly described. Simultaneously the pandemic has influenced depressive symptomatology by modifying economic, social and political structures, in addition to affecting daily routines. In both cases, associated immunological perturbations favoring a pro-inflammatory state could underlie an increased risk for depressive symptomatology. A resultant elevation in global depressive burden could further tax mental health care infrastructure and contribute to a range of worse health outcomes including diminished quality of life. This suggests a critical and time-sensitive need to better understand immune interfaces between depression and COVID-19. The unprecedented spread of SARS-CoV-2 has created a global emphasis on the immune system and its role in COVID-19 disease risk, outcomes and therapeutics . CytologPrior to SARS-CoV-2, depression was already a worldwide epidemic with considerable negative impact on morbidity and mortality. It is estimated that globally 350 million people are affected by depression, and it is a leading cause of disability , 11. In COVID-19 has the potential to induce widespread immunological effects as a direct result of infection and indirectly, independent of infection by modifying behavior and thought patterns. This may preferentially shift the immunological milieu toward an inflammatory state and predispose to higher rates of depressive symptomatology. The present review focuses on immune pathways linking COVID-19 infection with risk for depression as well as putative non-infectious immune mechanisms by which SARS-CoV-2 could increase depressive burden .As the data around short and long-term consequences of the COVID-19 pandemic accumulate, evidence suggests an urgent need to focus on neuropsychiatric sequelae. Though over 1,800 publications jointly mention COVID-19 and depression, fewer than 100 discuss the terms “immunity,” “depression” and “COVID-19.” Themes in these articles include: the role of complementary therapies, the beneficial role of physical activity, and pharmacological consideration and bidirectional interactions between COVID-19 and depression with a focus on stress, cytokine storm, and long-term depressive outcomes related to SARS-CoV-2 infection.The extant research includes mention of several complementary therapies purported to target shared immunological pathways in depression and COVID-19. These include the use of Ayurveda  traditioTwo recent reviews have focused specifically on molecular mechanisms linking immunity, COVID-19 and depression. In the first ) the autWorldwide, millions of cases of the infectious disease SARS-CoV-2 have been reported , accompaAcademic and public focus has also expanded to the long-term effect of SARS-CoV-2 infection on human health . This haFinally, attention has increasingly turned to the indirect ramifications of the virus. In globally disrupting routines, the economy, access to care and social dynamics, the pandemic could alter health outcomes for billions. Early data suggest these impacts may prove especially relevant for mental health. Health care workers managing COVID-19 patients in China reported increased psychological strain, including higher rates of depressive symptoms . A populEarly evidence that inflammation could precipitate depressive symptoms was derived from hepatitis patients receiving immunotherapy with interferon alpha. Many of these patients developed psychiatric symptoms including symptoms of depression . SubsequBrain alterations are central to the current understanding of depression pathophysiology . HoweverIn investigating the SARS-CoV-2 virus, research efforts have focused on the role of pre-existing immunological states as well as immune changes subsequent to infection. Direct correlations between COVID-19 outcomes and individual cytokines and immune cell populations indicate symptoms of COVID-19 are associated with elevations in interleukin 1 beta (IL-1β), IL-6, interleukin 10 (IL-10), and TNFα), as well as a general lymphopenia –49. IncrAn infection-related surge in proinflammatory mediators in COVID-19 has been called the cytokine storm or cytokine release syndrome. While consensus on the exact definition of the cytokine storm is debated, it is characterized by elevation in a range of immunological markers including interleukins, interferon-γ, TNF, chemokines and plasma proteins including complement and C-reactive protein (CRP) . In cytoAt this time, the precise methods by which the SARS-CoV-2 virus influences the CNS remain unclear. However, the widely cited symptoms of anosmia and dysgeusia suggest a high prevalence of CNS involvement , potentiOn entering the CNS, peripherally generated inflammatory mediators may amplify their effects on the brain by acting on microglial cells. Microglial activation represents a transition from a state of relative quiescence to a “primed” state in which the microglia increase production of cytokines and other inflammatory mediators . MicroglThose with existing neuroimmunological diseases may have heightened vulnerability to depressive symptomatology as a result of the COVID-19 pandemic. For example, people with multiple sclerosis demonstrated elevated rates of psychological distress including depression after easing of lockdown measures . ParkinsData demonstrating the long-term impact of COVID-19 on neuroimmune function remain limited. However, early survey results suggest that after acute infection, some experience residual symptoms of including fatigue, headache and anosmia, indicating a degree of persistent neurological alteration . Animal Neuroplasticity describes neural synaptic reorganization in response to environmental input. It is thought to form the basis for memory and learning . ImpaireImmune function is thought to exert a degree of control over neuroplasticity . This maAs regulators of CNS immunity, microglial cells are implicated in neuroplasticity. Microglia are thought to influence this process through glutamate homeostasis and production of inflammatory cytokines . On deteNeurogenesis is the process of creating new neurons. Once thought restricted to early life, human research now shows that neurogenesis occurs in discrete zones of the brain into adulthood, including the hippocampus and lateral ventricle , 77. LikNeuroplasticity and neurogenesis are affected by neurotrophic factors, compounds that bind to tyrosine kinase receptors and augment neuronal function, survival and development . Of thesImmunity plays a role in both BDNF expression and function. Administration of LPS reliably induces inflammation, including in the CNS . In animThe aforementioned pathway suggests a molecular mechanism by which infection with COVID-19 could directly downregulate levels of BDNF. However, non-infectious effects of the pandemic may also play a role, as chronic stress is thought to have a deleterious effect on BDNF expression . This imDecreased brain bioavailability of the tryptophan (TRP) metabolite serotonin (5-HT) underpins the psychopharmacology of the most commonly prescribed antidepressants . TRP met5-HT may directly influence immune homeostasis by suppressing Th17 differentiation, increasing expression of T regulatory cells (Tregs) and promoting M2-polarization of macrophages . These iMore robust research focuses on the effects of immunity on TRP metabolism, including 5-HT availability. Enzymatic action on the essential amino acid TRP determines whether it is converted in 5-HT or shunted into the kynurenine (KYN) pathway. In general, the majority of tryptophan enters the KYN pathway, creating downstream metabolites including KYN, kynurenic acid (KYNA) and quinolinic acid (QUIN) .Initiating enzymes in the KYN pathway are tryptophan 2,3-dioxygenase (TDO) and indolamine-2,3-dioxygenase (IDO). Notably, IDO is highly expressed in immune cells  and compThe relative increase in KYN pathway activation as a result of inflammatory immune activation has been proposed to contribute to depression through 5-HT depletion, though more recent focus has shifted to the differential neuroactive effects of KYN metabolites . For exaIn sum, an elevation in systemic inflammation as the direct result of infection with SARS-CoV-2 or as a result of pandemic-related behavioral changes and psychological stressors could predispose to relative brain 5-HT depletion and imbalance in KYN pathway metabolites that increase risk for depression .Increased psychological stress has been widely documented in response to COVID-19. Personal diagnosis of or the diagnosis of a close contact with COVID-19 have each been associated with elevated stress as well as with increased symptoms of depression . PoliticAlterations in stress pathways including the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS) are known risk factors for mental health conditions including anxiety and depression , 105. EaElevated psychological stress is proposed to induce immunological alterations through a variety of dose and duration-dependent mechanisms. Acute as well as chronic psychosocial stressors are associated with elevated inflammation . Acute sCompared with acute stressors, chronic stress may better predict the low-grade inflammatory immune activation correlated with depression . ProposeCOVID-19 related restrictions have sparked concern for decreased physical activity. In a recent survey of American adults, people active prior to COVID-19 restrictions reported a 32% reduction in physical activity . It is aPhysical activity has been shown to be an effective therapy for unipolar depression, with a benefit comparable to antidepressants and psychotherapy , 122. RePhysical activity is thought to positively affect multiple aspects of immune function, with a beneficial suppressive effect on inflammation . As exerEarly research during the COVID-19 pandemic suggests the potential for a negative impact on dietary patterns, potentially as a response to increased chronic stress and changes in habits , 130. FoIn both children and adults, data indicate a COVID-19-related increase in intake of prototypically inflammatory foods including refined carbohydrates and sugary drinks and in adults, a decreased intake of fish and fruit –135. ThiDietary influences on immune function are diverse. The role of macro and micronutrient deficiency in immune dysfunction is well-characterized, and dietary fiber, omega-3 fatty acids and polyphenols are also purported to play a role in healthier immune function . ConversAs a whole, these data suggest that changes in dietary patterns may represent a behavioral variable in inflammatory modulation, and that psychological stress-mediated trends during COVID-19 could have a detrimental effect on mood by increasing preference for less healthful foods. Emphasis on the consumption of a less processed diet and increased access to more nutrient-rich foods could represent a potential offset to diet-related immunological effects on depressive symptoms as a result of the pandemic.Depression and COVID-19 demonstrate shared patterns of immunological function, especially around a pro-inflammatory state characterized by elevation in cytokines including IL-6, TNFα, and IL-1β. SARS-CoV-2 may increase immunological risk for depression through direct infection-related influences on the CNS, or through associated behavioral shifts in diet, physical activity and psychological stress which subsequently promote an inflammatory immune state. Insight into the immunological intersections between depression and SARS-CoV-2 may help in the creation of strategies to mitigate of depression risk during the COVID-19 pandemic.The author confirms being the sole contributor of this work and has approved it for publication.AP receives consulting fees from Big Bold Health.


Guanidinoacetic acid (GAA) is a natural amino acid derivative that is well-recognized for its central role in the biosynthesis of creatine, an essential compound involved in cellular energy metabolism. GAA  has been investigated as an energy-boosting dietary supplement in humans for more than 70 years. GAA is suggested to effectively increase low levels of tissue creatine and improve clinical features of cardiometabolic and neurological diseases, with GAA often outcompeting traditional bioenergetics agents in maintaining ATP status during stress. This perhaps happens due to a favorable delivery of GAA through specific membrane transporters (such as SLC6A6 and SLC6A13), previously dismissed as un-targetable carriers by other therapeutics, including creatine. The promising effects of dietary GAA might be countered by side-effects and possible toxicity. Animal studies reported neurotoxic and pro-oxidant effects of GAA accumulation, with exogenous GAA also appearing to increase methylation demand and circulating homocysteine, implying a possible metabolic burden of GAA intervention. This mini-review summarizes GAA toxicity evidence in human nutrition and outlines functional GAA safety through benefit-risk assessment and multi-criteria decision analysis. N-amidinoglycine) belongs to the class of organic compounds known as alpha amino acids and derivatives. GAA  is produced endogenously in the human body from non-essential amino acids glycine and arginine, in a reaction controlled by an enzyme L-arginine:glycine amidinotransferase (AGAT) (2) from arginine to glycine to synthesize GAA, with ornithine as a byproduct. The reaction mainly takes place in the kidney, liver, and pancreas; however, GAA is also produced in the skeletal muscle, brain, and across the gut [S-adenosyl-L-methionine, a reaction catalyzed by guanidinoacetate N-methyltransferase (GAMT), to produce creatine and S-adenosyl-L-homocysteine. The formation of creatine is a major metabolic fate of GAA, and AGAT-driven reaction is considered a rate-limiting step of creatine biosynthesis [Guanidinoacetic acid  . AGAT ca the gut ,2,3. In ynthesis . Since cynthesis , the GAAynthesis ,7, previynthesis ); monocaynthesis .Plasma and urine GAA concentrations of 2.3 ± 0.8 μmol/L and 31.2 ± 21.7 mmol/mol of creatinine likely illustrate natural equilibrium in GAA metabolism . HoweverThe first documented report of GAA utilization as an experimental nutritional intervention in humans arguaby dates back approximately 70 years ago. The group of Henry Borsook from Caltech University demonstrated the beneficial effects of GAA (combined with betaine) in treating cardiac decompensation . The autAfter this inception, subsequent decades prompted a relatively limited interest in studying dietary GAA until 1999, when a Japanese group put forward GAA as a nutritional supplement to compensate for a disease-driven GAA shortage in patients with chronic renal failure . During S-adenosyl-L-methionine, an excessive GAA intake can hypothetically drain the stores of methyl donors in the human body . The metabolic burden of methyl donor deficiency can perturb many cellular functions, including DNA methylation, neurotransmission, antioxidant defense, and protein synthesis [Even the seminal paper that described the biochemical basis of GAA treatment recognized the possible risk of methyl group depletion following GAA consumption . Since tynthesis . Severalynthesis . We founynthesis , a critiS-adenosyl-l-homocysteine is a byproduct of GAA utilization that is further converted to homocysteine in a simple one-step reaction catalyzed by adenosylhomocysteinase: S-adenosyl-l-homocysteine + H2O <=> l-homocysteine + adenosine. Since hyperhomocysteinemia has been recognized as an independent risk factor for various cardiometabolic diseases [diseases , a possidiseases . Elevatediseases , with a diseases . In the diseases . This trdiseases . Similardiseases ,51. HaviSeveral in vitro and animal studies documented neurotoxic effects of exogenously administered GAA. The group of Angela Wyse reported that the intrastriatal injection of GAA induced inhibition of Na+/K+-ATPase activity , glutamaAn exposure to GAA in animal studies exacerbated ethanol-induced liver injury ,61, stimThe risk-benefit assessment is an emerging concept in the area of food safety ,66, withGAA is an investigational dietary supplement. Preliminary human studies suggest that dietary GAA has a relatively acceptable safety profile, yet medium-term intake appears to provoke unfavorable biochemical abnormalities, such as the rise in serum homocysteine (which could be attanuated by GAA co-ingested with creatine). Other adverse events demonstrated in animal studies with non-enteral administration of GAA are not confirmed in human trials with supplemental GAA thus far. Still, the possible toxic effects of GAA in the nervous system reported in pre-clinical research  remain of high concern, with additional human studies required before advancing GAA for human use. Whether beneficial effects outcompete side effects of GAA currently remains unknown in terms of evidence-based efficacy and safety data. A comparative analysis of supplemental GAA safety using multi-criteria decision analysis remains highly warranted to optimize treatment selection within the settings of nutrition and clinical bioenergetics.


Nursing home social workers (NH SW) at the frontline during COVID-19 are faced with many challenges in meeting the psychosocial needs of residents while balancing their own well-being needs. In order to explore the experiences of NH SW during COVID-19, the study utilized a cross-sectional survey distributed to social media sites  and professional networks. The survey asked participants (N = 63) open-ended questions which were analyzed using the rigorous and accelerated data reduction (RADaR) method. Themes suggested that fear for self, lack of administrative support, and overall stress were notable concerns among NH SW. Findings also suggested that support from family/friends and self-care were most personally helpful to NH SW. Lastly, themes related to coping strategies included talking with co-workers, mindfulness, and boundary setting. Findings suggest the need for increased supports for NH SW. Implications related to stress and coping during COVID-19 are offered.


Transgender Education for Affirmative and Competent HIV and Healthcare (TEACHH)’. This theoretically-informed community-developed intervention aimed to increase providers’ gender-affirming HIV prevention and care knowledge and competency and reduce negative attitudes and biases among providers towards trans women living with and/or affected by HIV.Transgender (trans) women face constrained access to gender-affirming HIV prevention and care. This is fueled in part by the convergence of limited trans knowledge and competency with anti-trans and HIV-related stigmas among social and healthcare providers. To advance gender-affirming HIV service delivery we implemented and evaluated ‘n = 78) participated in a non-randomized multi-site pilot study evaluating TEACHH with a pre-post-test design. Pre- and post-intervention surveys assessed participant characteristics, intervention feasibility  and acceptability . Paired sample t-tests were conducted to assess pre-post intervention differences in perceived competency, attitudes/biases, and knowledge to provide gender-affirming HIV care to trans women living with HIV and trans persons.Healthcare and social service providers and providers in-training  working with trans women living with and/or affected by HIV , and 2) knowledge, attitudes/biases and perceived competency in gender-affirming healthcare . Greater change in outcome measures from pre- to post-intervention was experienced by those with fewer trans and transfeminine clients served in the past year, in indirect service roles, and having received less prior training.The intervention was feasible (100% workshop completion) and acceptable (91.9% indicated interest in future gender-affirming HIV care trainings). Post-intervention scores indicated significant improvement in: 1) knowledge, attitudes/biases and perceived competency in gender-affirming HIV care (score mean difference (MD) 8.49 . Barriers to gender-affirming HIV prevention and care for transgender (trans) women – a diverse group of people assigned male at birth who typically identify as women, trans women, and/or transfeminine – are an enduring health equity issue for those living with and affected by HIV. These barriers contribute to disproportionately high rates of HIV in comparison to cisgender (cis) adults (those whose gender identity and sex assigned at birth are congruent) . A recenAccess to HIV prevention, treatment, and support are critical to realizing optimal health and wellbeing among trans women , 4. ThusWell-documented barriers to trans women’s access to HIV prevention and care are stigmas related to trans identity, HIV, and other characteristics, perpetuated by healthcare and social service providers –22. For gender-affirming care when they: 1) have foundational knowledge about issues associated with trans people in addition to understanding appropriate terminology; 2) reconcile personal beliefs, biases and attitudes towards trans people with their professional role; 3) actively work towards creating an inclusive healthcare environment; and 4) support patients in defining their gender and their relationship with gender ) Notably, only four participants indicated having received any trans-specific training in a formal college/university-level course. This is consistent with a plethora of research documenting low hours of LGBTQ content in medical and allied health professional training , 27. OthAdditionally, allied healthcare professional education could take a regional approach and seek to foster competent and affirming care in rural settings . For exaTrans women face many barriers to accessing HIV prevention and care, notably lack of knowledge and competency as well as trans-related and HIV-related stigma among their providers –22. The


Inflammation has proven to be a key contributing factor to the pathogenesis of ischemic and hemorrhagic stroke. This sequential and progressive response, marked by proliferation of resident immune cells and recruitment of peripheral immune populations, results in increased oxidative stress, and neuronal cell death. Therapeutics aimed at quelling various stages of this post-stroke inflammatory response have shown promise recently, one of which being differentiated induced pluripotent stem cells (iPSCs). While direct repopulation of damaged tissues and enhanced neurogenesis are hypothesized to encompass some of the therapeutic potential of iPSCs, recent evidence has demonstrated a substantial paracrine effect on neuroinflammation. Specifically, investigation of iPSCs, iPSC-neural progenitor cells (iPSC-NPCs), and iPSC-neuroepithelial like stem cells (iPSC-lt-NESC) has demonstrated significant immunomodulation of proinflammatory signaling and endogenous inflammatory cell populations, such as microglia. This review aims to examine the mechanisms by which iPSCs mediate neuroinflammation in the post-stroke environment, as well as delineate avenues for further investigation. Stroke remains a leading cause of death and long-term neurological disability in the aging population worldwide. Despite recent therapeutic efforts and improved control of stroke risk factors, the mean global lifetime risk of stroke has increased over the past 20 years. Historically, this disease has predominantly affected older adults, with 36.6% of individuals over 60 experiencing a stroke . There iCurrent FDA-approved AIS therapeutics are primarily aimed at recanalization of the ischemic vessel through thrombolytic or endovascular means . While tInflammation has proven to be a key contributing factor to the pathogenesis of ischemic and hemorrhagic stroke , represeiPSCs are a viable regenerative therapeutic in a range of degenerative conditions. These cells, which can be induced from somatic cells to pluripotent stem cells , allow nRodent middle cerebral artery occlusion (MCAO) models of stroke have served as a means in which to test the anti-inflammatory potential of iPSCs. While some paradigms utilize a permanent occlusion, others invoke transient occlusions ranging from 30- to 90- min . This ocIn contrast to this ischemic stroke study, other studies suggest a less defined role of iPSCs in the stroke environment. One rodent dMCAO model of stroke found a significantly increased expression of SDF-1/CXCL12  in the peri-infarct area of iPSC-treated animals above that of non-stroked animals. No significant difference, however, was observed in the expression of SDF-1 between stroked non-treated animals and iPSC-treated animals . While SOutside of ischemic models of stroke, investigation of the anti-inflammatory potential of iPSCs has been conducted in intracranial hemorrhagic (ICH)  models o+  and CD11b+  cells, as well as a decreased expression of activated Caspase-3+ (cellular apoptosis)/NeuN+  cells. Lastly, Nissl (nucleic acids) staining at day 42 post-ICH revealed less damage to Nissl bodies and nuclei within neural cells in the iPSC-treated group than the PBS (vehicle) group, and significantly decreased GFAP+ expression at day 42 post-ICH was detected in iPSC-treated tissue  model was conducted in which iPSC-NSCs were arranged in a membrane-separated co-culture system with OGD cortical cells (+ (deoxynucleotidyl transferase dUTP nick end labeling) and GFAP+ cells as well as significant increases in residual neurons and neurites of iPSC-NSC groups compared to OGD cells alone. Interestingly, this pattern of responses was also seen in iPSC groups but was not observed in bone marrow-derived mesenchymal stem cells (BM-MSCs) or Wharton’s jelly MSC groups. Therefore, these antiapoptotic and anti-inflammatory responses seems to be specific to iPSCs and iPSC-NSCs. Messenger RNA expression level analysis revealed a significant increase in BMP7 (bone morphogenic protein 7), CXCL14 , FGF12 , and FGF8  over iPSCs and MSCs, as well as FGF9 , and IGFBP2 (insulin-like growth factor protein) over MSCs. While most of these factors have known implications in neuronal development and NSC differentiation, factors such as BMP7, CXCL14, and IGFBP2 have been known to play a role in anti- and proinflammatory processes as well .Cytokine array analysis of culture supernatants also revealed a significant enrichment of IGFBP2 in iPSC-NSCs over BM-MSCs and OGD-injured cultures. When these cytokines  were individually dosed onto OGD cells, a significant neuroprotective and anti-inflammatory phenotype was observed upon immunocytochemical quantification—similar to that observed with iPSC-NSC co-culture. Specificity of these factors to carry out mechanisms of neuroprotection and anti-inflammation was confirmed by a neutralizing antibody cocktail which abolished these effects . Lastly,in vivo conditions, grafted iPSC-NSCs in ECM form rosettes, develop radial glia, and are vascularized by 8 weeks post-engraftment. Interestingly, while cell-free ECM grafts were found to have reactive microglia accumulating at the boundary of the graft, only quiescent host microglia were found within the organoid structures of the iPSC-NSC + ECM group 4 weeks post-engraftment. These results suggest iPSC-NSCs promote an anti-inflammatory environment in vivo, which is not contingent on an ischemic or hemorrhagic condition, and that they protect against chronic innate immune cell activation following trauma (Extracellular matrices (ECM) can also enhance the anti-inflammatory potential of iPSC-NSCs. In non-stroke g trauma .in vivo rodent MCAO models revealed significant decreases in TNF-α, IL-6, IL-1β, intercellular adhesion molecule 1 (ICAM-1), vascular cells adhesion molecule (VCAM-1), monocyte chemoattractant protein (MCP-1), and macrophage inflammatory protein (MIP-α) expression of iPSC-NSC-treated animals compared to non-treated animals. Additional protein level analysis revealed a significant decrease in mouse immunoglobulin G (IgG) and matrix metallopeptidase 9 (MMP9) (Gene expression data in 9 (MMP9) . MMP9 is9 (MMP9) . The acc9 (MMP9) . More woin vivo MCAO models of ischemic stroke have also found similar anti-inflammatory and neuroregenerative potential of iPSC-NSCs. Studies in rodent models have found significant reductions in IBA1+ (+ (+ cells (+ and GFAP+ cells in the acute and chronic stroke environment administered the therapeutic at subacute time points (∼7 days post-MCAO), the latter study administered cells at an acute (24 h) timepoint. This difference could have implications for the therapeutic window of iPSC-NSCs to exert their effects.Immunocytochemistry data of in IBA1+ , ED1+ (CIBA1+ (+ , and GFA(+ cells  in the i(+ cells  as far a(+ cells . Interes+ area in iPSC-NSC-treated animals compared to non-treated animals. There was not a significant reduction in GFAP+ cells however (To assess the translational potential of iPSC-NSCs as a clinical stroke therapeutic, their anti-inflammatory efficacy in large animal models of stroke must also be considered. The increased complexity, size, and cellular organization of large animal models serve to assess scaling, dosing, or diffusion-limited processes. Additionally, due to a white-to-gray matter composition which more closely resembles humans, differential effects of iPSC-NSCs on these cells can be assessed . In a po however . While tSimilar to iPSC-NSCs, neuroepithelial-like stem cells have also been differentiated from iPSCs and are distinct from radial glia-like NSCs . The effOutside of the lineages discussed in this review, there are many iPSC-derived lineages which exhibit noted anti-inflammatory effects but have not yet been investigated in the context of stroke. For example, iPSCs-MSCs  have beein vitro culture expansion and reprogramming, has remained a major hurdle to clinical implementation. This complex discussion involves consideration of the implications of single nucleotide variants, mutations in non-coding regions, and mutations in recognized cancer genes. Whether these mutations would lead to cancerous proliferation or affect the therapeutic potential of iPSC cells remains a topic of active debate (While the functional effects of iPSCs and iPSC-derived cells in stroke are clear—decreasing lesion volume  and morte debate  and warre debate  and inhie debate . AssessmSS and DH designed, wrote, and edited the manuscript and have approved it for publication. Both authors contributed to the article and approved the submitted version.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.


CircRNAs (circular RNAs) are single-stranded RNAs that form covalently closed loops and function as important regulatory elements of the genome through multiple mechanisms. Increasing evidence had indicated that circRNAs, which might serve as either oncogenes or tumor suppressors, played vital roles in the pathophysiology of human diseases, especially in tumorigenesis and progression. CircRNA-ZFR (circular RNA zinc finger RNA binding protein) is a circular RNA that had attracted much attention in recent years. It has been found that circRNA-ZFR was abnormally expressed in a variety of malignant tumors, and its dysregulated expression was closely related to tumor stage, cancer metastasis and patients’ prognosis. Recent studies had shown that aberrantly expressed circRNA-ZFR could regulate the malignant biological behaviors of tumors through various mechanisms; further exploration of circRNA-ZFR expression in tumors and its regulation on malignant biological behaviors such as tumor proliferation, invasion and drug resistance will provide new ideas for clinical tumors diagnosis and treatment. The genome-wide studies showed that greater than 70% of the human genome is transcribed into RNAs, while only approximately 2% of the sequences have the capacity to encode proteins. The non-coding RNAs (ncRNAs), which were long assumed to be transcriptional noise, comprise the most proportion of the transcripts Covalently closed circRNAs (circular RNAs) were originally identified in plant viroids, yeast mitochondrial RNAs, and hepatitis virus . CircRNACircRNA-ZFR (zinc finger RNA binding protein) is a new circRNA (circBase ID: hsa_circ_0072088), which had been confirmed to be located on human chromosome 5p13.3 Shown in . As a meBreast cancer (BC) is one of the most common gynecological cancers. The deteriorating environment and lifestyle flaws are raising the frequency of this cancer . The curα) as a functional target of miR-578 in regulating BC cell viability, migration, invasion, glycolysis and anti-apoptosis. In BC tissues and cell lines, HIF1A mRNA expression was positively correlated with circRNA-ZFR expression and negatively correlated with miR-578 expression. The results of luciferase report assay and RNA pull-down indicated that circRNA-ZFR modulated HIF1A expression via acting as a sponge of miR-578. These findings suggested that the aberrant expression of circRNA-ZFR in breast cancer promoted the tumor proliferation, invasion, migration and inhibited cell apoptosis by regulating miR-578/HIF1A axis.By analyzing the Gene Expression Omnibus (GEO) dataset, β-catenin and PI3K/AKT pathways were the downstream effectors of Met; both signaling pathways were in manage of multiple biological processes and frequently aberrantly activated in human cancers  tissues as compared with para-carcinoma tissues, and circRNA-ZFR expression was also notably raised in CAKI-1, ACHN, A498 and KTCTL-26 cells.  cancers . To detein vivo and in vitro; and as shown in the bioinformatics analysis, circRNA-ZFR was predicted to possess binding sites for miR-1270 and miR-545. The western blot and qRT-PCR detection results indicated that miR-1270 and miR-545 expression was significantly elevated in both T24 and J82 cells when circRNA-ZFR was attenuated. The results from RNA pull-down and dual-luciferase reporter assay also proved that circRNA-ZFR could directly interact with miR-545 and miR-1270. Otherwise, the probability of WNT5A (Wnt family member 5A) binding with miR-545 and miR-1270 was predicted by starBase, and these targeting relationships were verified by luciferase reporter assay. Previous studies had found that Wnt family genes played a vital part in human organogenesis and tumor genesis through regulating WNT/β-catenin signaling stimulate  was significantly reduced by the attenuation of circRNA-ZFR, which was partially rescued by the application of miR-1270/545 inhibitors or WNT5A overexpression. Interestingly, timulate . In addiCC  is one of the most common gynecological cancers in women all over the world. In the early stages, HPV-associated CC development is asymptomatic and its high invasiveness and mortality threaten the health of more and more women . In the By comparing the circRNA-ZFR expression between NSCLC  tumor tissues and matching nontumor tissues of 45 patients, Besides, the study of in vivo and in vitro.Currently, the treatment strategies for NSCLC include surgery, radiotherapy, chemotherapy and immunotherapy. Drug resistance in patients with NSCLC in the context of chemotherapy remains a major barrier to the treatment of NSCLC . CircRNAβ-catenin pathway. Whereas, β-catenin signal pathway, which is closely linked to HCC cell proliferation and migration. The results of Yang’s research implied that circRNA-ZFR and AKT1 expressions were up-regulated and miR-511 expression was down-regulated in hepatocellular carcinoma. What’s more, circRNA-ZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circRNA-ZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, and activated cascades of proliferation-related proteins . These data indicated that circRNA-ZFR might promote cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma.As an important component of MAP kinase signal transduction pathway, MAP2K1 (mitogen-activated protein kinase kinase 1) has been proved to be involved in the regulation of cell proliferation, differentiation and gene transcription . Cedric’via venn diagram, and the intersection was PTEN (phosphatase and tensin homolog). Moreover, The RIP and dual-luciferase reporter assay results indicated that circRNA-ZFR could target miR-107/miR-130a and PTEN was the downstream target gene of miR-107/miR-130a. The CCK-8 assay and flow cytometry analysis results displayed that circ-ZFR influenced GC cell propagation, cell cycle and apoptosis resistance by miR-107/miR-130a/PTEN axis. Interestingly, the rescue experiment results showed PTEN overexpression also attenuated the effects of circRNA-ZFR silence in GC cells, and the xenograft mice model experiment results showed that circRNA-ZFR curbed GC tumor growth and affected p53 protein expression in vivo. In general, circRNA-ZFR inhibited cell proliferation and promoted apoptosis in GC by miR-130a/miR-107/PTEN axis. By analyzing the circRNA-ZFR expression in 30 colorectal cancer tissues and matched non-tumor normal tissue specimens, The qRT-PCR detection results showed that circ-ZFR was drastically downregulated in gastric cancer tissues and cells. As confirmed by CCK-8 assay, overexpression of circ-ZFR observably suppressed GC (gastric cancer) cell proliferation; flow cytometry analysis disclosed that the apoptosis rate of GC cells was significantly increased after circRNA-ZFR overexpression, and the proportion of cells staying in G1/G0 phase significantly increased. Through starBase, a bioinformatics prediction website, With the research development, the aberrant expression of circRNAs has been gradually recognized as a hallmark feature in cancer. Investigating these molecules as biomarkers or therapeutic targets will be a promising field for cancer treatment . Numerou


Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths worldwide. Unlike other types of cancer, HCC can be treated with locoregional treatments (LRTs) such as radiofrequency ablation (RFA) or transarterial chemoembolization (TACE). However, recurrences following LRTs are common, and strategies to improve long-term outcomes need to be developed. The exhaustion of anti-tumor immunity in HCC has been well established in many reports and the immunomodulatory effects of LRTs  have also been reported in several previous studies. However, a comprehensive review of previous studies and the possible roles of immunotherapy following LRTs in HCC are not known. In this review, we discuss the immunological evidence of current clinical trials using LRTs and combined immunotherapies, and the possible role of this strategy. Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related deaths worldwide, and its incidence is increasing despite recent advances in treatment and surveillance strategies for chronic liver diseases, including viral hepatitis . AlthougLRTs have a substantial recurrence rate; the median overall survival (OS) was 60 months and a 50–70% recurrence rate after successful RFA within 20–30 months has been reported . FurtherSince its approval in 2007, sorafenib has been the only option for the systemic treatment of advanced HCC. Other systemic treatments have recently been developed for advanced stage, including novel tyrosine kinase inhibitors (TKIs) such as lenvatinib as a first-line treatment and regorafenib or cabozantinib as second-line treatment. Therefore, a combination of TKIs and LRTs has been investigated to improve clinical outcomes. However, sorafenib followed by resection or ablation  has faiImmune checkpoint blockade (ICB), such as nivolumab is indicated for unresectable HCC. Nevertheless, monotherapies using ICBs failed to show a marked effect in HCC compared to other tumors, as shown in the recent clinical trials . The livA previous report suggested that the recent advancement in the surveillance and treatment modalities during the 15 years has led to early detection of HCC, improved treatment efficacies, and prolonged patients’ survival . These fIn this manuscript, we briefly review the immunological characteristics of HCC, including rationales for ICBs, and summarize the changes after each LRT. Based on these summaries, current trials on adjuvant immunotherapies following LRTs in HCC are described.+ T cells, which are the main effectors of anti-tumor adaptive immune responses, upregulate immune checkpoint molecules such as PD-1, in addition to CTLA-4, hepatitis A virus cellular receptor 2 (TIM-3), and lymphocyte activating gene 3 (LAG-3) in patients with HCC, and blocking them in vitro significantly restored the function of exhausted CD8+ T cells [high signatures had better median OS in the TCGA cohort [+ T cell responses, thereby augmenting the adaptive immune responses against tumors. Based on this evidence, compared with sorafenib, nivolumab in patients with advanced HCC as a first-line treatment tended to improve survival [Currently available ICBs are anti-PD-1 (nivolumab), anti-PD-1 plus anti-CTLA-4 (pembrolizumab plus ipilimumab), and anti-PD-L1 (atezolizumab) combined with anti-vascular endothelial growth factor (VEGF) bevacizumab . CD8+ T  T cells ,22. Furt T cells . For exaA cohort . FurtherA cohort . Therefosurvival  in a pha+ macrophages were associated with poor survival [+IL-10+ B cells suppress anti-tumor CD8+ T cell responses [In addition, PD-L1-expressing HLA-DRsurvival  and PD-Lesponses . Furtheresponses . T cellsesponses ,29, and esponses . Therefoesponses , which m+ T cells, other immune cells contribute to weak anti-tumor CD8+ T cell responses in HCC. CD4+CD25+FoxP3+ regulatory T (Treg) cells exhibit on their surface both CTLA-4 and PD-1 and can be a target cell population of ICBs [+ T cell-responses [+ dendritic cells (DCs) are also associated with the attenuation of anti-tumor T cell responses due to indoleamine 2, 3-dioxygenase (IDO) and IL-10 production [In addition to chronic antigenic stimulation and exhaustion of CD8 of ICBs . They aresponses ,33. Intresponses . Among vesponses . Therefooduction . As exhaThe results of previous basic and clinical studies indicate that a better understanding of the immunosuppressive environment in HCC may provide novel therapeutic strategies for advanced stage HCC. However, adjuvant treatment following LRT or surgical treatment has not been established in early to intermediate stage HCC. The promising results of ICBs in patients with advanced HCC indicate possible options for adjuvant treatment in early to intermediate stage HCC. In melanoma, ICBs have been studied and approved as adjuvant treatments. The use of adjuvant pembrolizumab in stage III resected patients  and nivoRecently, the synergistic effect of systemic therapy and ICBs has been actively reported. VEGF/VEGFR signaling is associated with the immunosuppressive tumor microenvironment, in addition to its primary role in angiogenesis . For exaCurrently available TKIs also have immunomodulatory effects. Firstly, used TKI sorafenib has been studied for its various impacts on immune cells. It can improve anti-tumor immune responses by regulating macrophages  and MDSCSeveral mechanisms have been suggested regarding the effects of LRTs on anti-tumor immune responses; cancer cell death caused by LRTs is closely associated with immunomodulatory effects. Both apoptosis and necrosis of tumor cells can be induced by LRTs. In general, necrosis is known to be immunogenic, because the plasma membrane is broken, and necrotic cells release cellular contents including cancer-specific antigens and DAMPs . This phThe release of tumor antigens due to cell death and subsequent changes in antigen-presenting cells (APCs) and effector immune cells are the main processes responsible for the changes in anti-tumor immune responses after LRTs . Tumor c+ T cell responses and was associated with the release of peptidases [The type of cell death might also be important in inducing an anti-tumor immune response. In a previous study, the apoptosis of tumor cells due to irradiation (not injection) of necrotic tumor cells augmented the effects of tumor cell vaccines . In anotptidases . LRTs inptidases . In anotptidases . Most ofptidases . These fDoxorubicin, which is the most commonly used agent in TACE, can induce immunogenic cell death. The effects on cell death can differ with the types of chemotherapeutics; doxorubicin can promote immunologic cell death, but cisplatin cannot . Convers+-exhausted CD8+ T cells within the tumor were also increased in patients with stage I and stage II or III HCC [+ T cells exhibit exhausted features [Evidence regarding immunological changes based on the HCC stage is lacking; however, in the analysis of the exhausted, dysfunctional immune cells within the tumor and adjacent liver, the surgical samples used in most immunological studies were from relatively early stage HCC patients. Immune dysfunction may begin in the early stage of tumor development. In a murine model, tumor-specific T cell exhaustion was initiated at the early pre-malignant phase of tumorigenesis . In a pr III HCC . Furtherfeatures . Althougfeatures . Thus, iRFA is the most studied LRT in terms of immunological changes following LRTs. The immunomodulatory effect of RFA may be associated with tumor cell death caused by coagulative necrosis and apoptosis . CryoablThe increased number of tumor-specific T cells is associated with reduced tumor recurrence following RFA . SimilarA 50–70% recurrence rate after successful RFA within 20–30 months has been reported in several studies . In a pr+cells in a CCR1-dependent manner [The Toll-like receptor 9 agonist CpG increased anti-tumor T cell responses following RFA and prolonged survival and reduced new tumor growth . A weak t manner . In addit manner . These f+-exhausted CD8+ T cells in the tumor were more increased in stage II or III patients than in stage I patients [TACE is the preferred method for treating intermediate-stage, unresectable HCCs. Despite procedural advancement, the clinical outcome has not been satisfactory and the median OS, which is affected by tumor size, tumor markers, liver function, and vascular invasion, was 19.9 months based on a recent report . Similarpatients , indicatpatients  and clinThe changes in the composition or function of immune cells after TACE remain to be elucidated, and whether the peripheral T cell population might be affected by TACE has been investigated in only a few studies. Growing evidence indicates that tumor necrosis caused by TACE induces immunological activation. For example, the inflammatory cytokine IL-6 significantly increased early after TACE . However+/CD8+ T cell ratio was significantly lower in patients with HCC prior to TACE than in healthy volunteers and was increased 1 month after TACE [+ T cells, which was associated with improved clinical outcomes [Immunologic cell death markers, such as high mobility group box 1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE), inducers of PD-L1 in tumor cells, are increased by TACE in HCC patients ,87. In ater TACE ,89, althoutcomes . Notablyoutcomes . Additiooutcomes . These fSeveral studies have indicated that TACE also affects Treg cells. Peripheral Treg cells were significantly increased in HCC patients and decreased by TACE ,92; theiTransarterial radioembolization (TARE) using yttrium-90 (Y90) is an emerging option for treating locally advanced HCC that is not eligible for surgical resection. The method delivers Y90 via tumor-supplying arteries and provides tumor-restricting effects without damaging the non-malignant liver , althoug+ T cells, CD56+ NK cells, and CD8+CD56+ NKT cells [+TIM-3+CD8+ T cells were observed in TARE responders, indicating that further enhancement of T cell responses might be feasible by using ICBs following TARE.A recent in-depth analysis using CyTOF in resected HCC samples who underwent TARE before resection showed that TARE induces the activation of local immune cells, including CD8KT cells . NotablyKT cells . FurtherBased on previous observations, several pilot studies have evaluated the possibility of LRTs combined with ICBs in patients with HCC. In a single-center retrospective study, LRTs, including TACE or TARE combined with nivolumab, were tolerated in a small number of patients with intermediate or advanced HCC without deterioration of liver function , indicatICBs as adjuvant therapies following LRTs are currently being investigated in phase III trials. Anti-PD-L1 combined with anti-VEGF agents might also be a promising strategy as adjuvant therapy following LRTs. Durvalumab (anti-PD-L1) plus bevacizumab (anti-VEGF) following TACE (EMERALD-1) or curative treatments including RFA and surgical resection (EMERALD-2) is being evaluated as a phase III, randomized, double-blind, placebo-controlled, multicenter trial. Primary endpoints are PFS (EMELALD-1) or recurrence-free survival (EMERALD-2). The estimated primary completion year would be 2022 for EMERALD-1 and 2023 for EMERALD-2. Atezolizumab plus bevacizumab is also under the open-label, randomized, phase III trial in patients who underwent curative treatments (IMbrave050), and the estimated primary completion year would be 2023. Furthermore, safety and efficacy (PFS and OS) of pembrolizumab plus multikinase inhibitor lenvatinib following TACE is currently being investigated (LEAP-012), and the estimated primary completion year would be 2025. Furthermore, combined regimen, nivolumab plus ipilimumab is also being investigated in an adjuvant setting following TACE (CheckMate 74W), and the primary endpoint is time-to-progression. The estimated primary completion year would be 2026. Combined LRTs and ICBs could be a breakthrough for treating HCC and innovative improvement in the prognosis of HCC patients.Taken together, the evidence indicates that LRTs can modulate the anti-tumor immune responses in HCC patients, and the combination of LRTs and ICBs might have a synergistic effect on anti-tumor immune responses and clinical outcomes. Technically successful LRTs remain the most important because incomplete LRTs can induce angiogenesis in residual tumor cells . SeveralNovel ICBs such as anti-TIM-3, anti-TIGIT, or anti-LAG-3 were also shown to restore the function of tumor-infiltrating T cells in vitro , and the


Background Severe disease due to the novel coronavirus disease 2019 (COVID-19) has been shown to be associated with hypercoagulation. The aim of this study was to assess the Rotational Thromboelastometry (ROTEM) as a marker of coagulopathy in hospitalized COVID-19 patients.Methods This was a prospective, observational study where patients hospitalized due to a COVID-19 infection were eligible for inclusion. Conventional coagulation tests and ROTEM were taken after hospital admission, and patients were followed for 30 days. A prediction model, including variables ROTEM EXTEM-MCF (Maximum Clot Firmness) which in previous data has been suggested a suitable marker of hypercoagulation, age, and respiratory frequency, was developed using logistic regression to evaluate the probability of death.Results Out of the 141 patients included, 18 (13%) died within 30 days. In the final prediction model, the risk of death within 30 days for a patient hospitalized due to COVID-19 was increased with increased EXTEM-MCF, age, and respiratory frequency. Longitudinal ROTEM data in the severely ill subpopulation showed enhanced hypercoagulation. In an in vitro analysis, no heparin effect on EXTEM–coagulation time (CT) was observed, supporting a severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) effect on prolonged initiation of coagulation.Conclusion Here, we show that hypercoagulation measured with ROTEM predicts 30-day mortality in COVID-19. Longitudinal ROTEM data strengthen the hypothesis of hypercoagulation as a driver of severe disease in COVID-19. Thus, ROTEM may be a useful tool to assess disease severity in COVID-19 and could potentially guide anticoagulation therapy. The global emergence of the novel corona virus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has evolved rapidly achieving pandemic proportions with dire consequences for human health and welfare.Recent reports indicate a high incidence of thrombotic events in COVID-19 patients treated in intensive care units (ICUs),Elevated levels of fibrin degradation products  have consistently been suggested as a strong prognostic factor associated with poor outcome,Previous studies assessing ROTEM in critically ill COVID-19 patients suggest a procoagulant stateSeveral risk stratification tools referring to patients with COVID-19 across different settings and populations have been reportedIn this study, we aimed to evaluate several markers of coagulopathy in patients hospitalized due to COVID-19. Specifically, we intended to:Develop and test a pragmatic risk stratification score model, using ROTEM data in combination with other known risk factors to predict 30-day mortality.Assess the longitudinal course of ROTEM test results in severe disease.Examine the low molecular weight heparin (LMWH) effect detected by ROTEM, analyzed in an in vitro experiment.Evaluate the D-dimer-to-P-fibrinogen ratio as a marker of thrombotic activity.The study was a prospective, observational single-center study. Inclusion criteria were hospitalization due to verified COVID-19 infection and age over 18 years. No exclusion criteria were set up. After inclusion, a blood sample was taken and ROTEM analyzed, apart from this nothing differed from the standard care. The ROTEM analyses in this study were performed for research purpose only and ROTEM test results, as opposed to other laboratory test results, were not available for the treating physician and did therefore not affect treatment. All patients were followed-up after 30 days when outcomes were registered. The study was approved by the Swedish Ethical Review Authority (D-nr 2020–01875). In this ethical approval, consent was waived in very severe cases of COVID-19 disease where patients, due to medical conditions, were not able to give their consent.All patients enrolled in the study were included at Capio St Göran's Hospital, Stockholm, Sweden, during a 4-month recruitment period (May–August 2020). The diagnosis of COVID-19 was defined as either positive polymerase chain reaction (PCR) test for SARS-CoV-2, or as typical radiologic findings on chest computed tomography (CT), corresponding to COVID-19 classification score (CO-RADS) 4 or 5.In the statistical model, the ROTEM variable EXTEM-MCF (Maximum Clot Firmness) was deemed the most suitable candidate as a marker of hypercoagulation, based on a previously published analysis of a subset of the data.Comorbidity in the statistical model was defined as a prior diagnosis of either hypertension, diabetes, chronic obstructive pulmonary disease (COPD)/asthma, or cardiovascular disease.Previous thromboembolic disease among included patients was defined as a diagnosis of either arterial or venous thrombosis, registered in the medical journal at any time, prior to presentation of COVID-19 symptoms.ROTEM and CCTs , the International Normalized Ratio (INR), antithrombin, and platelet count) were collected after admission : 1–3]). Apart from these blood tests, nothing in the COVID-19 standard care of included patients was changed including ventilation strategies, medications, or routine examinations. To examine the longitudinal course of ROTEM in more severely ill patients, we performed repeated testing at days 5 and 10 after the first blood test. Patients who were discharged from hospital care prior to the second or third test were not tested. This longitudinal sample will therefore represent the development over time in cases with a more severe disease course. Furthermore, the D-dimer-to-P-fibrinogen ratio was calculated as a marker of thrombotic activity.During the study period, anticoagulant therapy was standard of care in COVID-19 pneumonia at Capio St Göran's hospital and was prescribed according to disease severity and thromboembolic risk profile. Routine anticoagulant treatment administrated after admission was the LMWH Tinzaparin  classified after dose regime . Patients with severe disease requiring ventilation support received high prophylaxis doses of antithrombotic treatment, and in case of verified or suspected thromboembolic disease, treatment doses of LMWH were used. Patients in regular wards without risk factors of thromboembolic complications received low-dose prophylaxis.ROTEM is an established point-of-care device, used for detecting and monitoring coagulopathy, providing rapid assessment of clot formation to lysis. A ROTEM sigma  was used for thromboelastometric analyses. Here, we present four ROTEM-variables as follows: (1) extrinsically activated assays with tissue factor (EXTEM), (2); intrinsically activated assays using phospholipid and ellagic acid (INTEM), (3) fibrin-based extrinsically activated assays with tissue factor and platelet inhibitor cytochalasin D (FIBTEM), and (4) intrinsically activated assays with the addition of heparinase (HEPTEM). EXTEM and INTEM test the extrinsic and intrinsic pathways, respectively. FIBTEM provides information of fibrinogen function, eliminating platelet contribution to clot formation. HEPTEM eliminates heparin effects. Within every ROTEM-variable, five parameters were quantified: CT which is the time (in seconds) from test start until an amplitude of 2 mm is reached, giving information about coagulation activation/initiation. Clot formation time (CFT) corresponds to the time (in seconds) between 2- and 20-mm amplitudes, giving information about clot propagation. Maximum clot firmness (MCF) is the maximum amplitude (in mm) reached during the test, giving information about clot stability. Lysis index (LI) 30 and 60 are the reduction in MCF 30 and 60 minutes after CT, respectively (in percent).n = 3), and we applied a repeated measures of analysis of variance (ANOVA).A prolonged EXTEM-CT, short EXTEM-CFT, and an increased EXTEM-MCF and/or FIBTEM-MCF suggest a hypercoagulable state with a prolonged initiation of coagulation. A prolonged INTEM-CT compared with HEPTEM-CT illustrates a heparin effect. To test the effects of different doses of tinzaparin on EXTEM-CT in vitro, four different concentrations of tinzaparin were added to blood from healthy donors  and R, version 3.6.1 was used for statistical analysis and visualizations.Categorical variables were introduced in the regression model by means of dummy variables. Numeric covariates were transformed with the most suitable power transformation. The transformed variables were entered to the predictive models through natural cubic splines when significant departures from linearity were detected. The choice of number and location of the knots were based on visual assessment and the Akeike's information criterion, respectively. The predictive properties of the model were evaluated by calculating the area under the curve (AUC) for the receiver operating characteristic (ROC) curve. Sensitivity, specificity, and positive and negative predictive values were calculated. All continuous variables were presented as median and IQR. Two-sided Wilcoxon's test was used to test for difference between groups for continuous variables and Fisher's exact test for categorical data. In the longitudinal data analysis, we used two-sided, paired Wilcoxon's test for the ROTEM variables EXTEM-CT, -MCF, and -CFT, respectively.This study was conducted and reported applying the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.A total of 141 COVID-19 patients with a median age of 63 years [IQR: 51–75] were included in the study, 87 (62%) were male . ComorbDemographic and clinical baseline characteristics for all patients were divided in survivors and nonsurvivors . EighteAmong nonsurvivors, 9 of 18 patients (50%) had ongoing anticoagulant or platelet inhibiting treatment before hospital admission, prescribed prior to their SARS-CoV-2 infection . Ten (56%) of nonsurvivors had cardiovascular disease . Among survivors, 16 of 123 (13%) had anticoagulant treatment prescribed before inclusion and 19 (15%) had a history of cardiovascular disease.p = 0.01 and 0.002). Platelet count, INR, P-fibrinogen, and antithrombin did not differ significantly among survivors compared with nonsurvivors. EXTEM-/INTEM-CT were significantly prolonged in non-survivors compared with survivors. EXTEM-/FIBTEM-MCF were in upper reference ranges and EXTEM-/INTEM-LI30 and -LI60 indicated low fibrinolytic activity in survivors, as well as nonsurvivors.Laboratory test results are presented inThe final logistic regression prediction model:Logit(death) = −9.6 + (9.5) * MCF + (0.15) * age + (−155) * RFLogit(death) is the predicted log odds of 30-day mortality (here synonymous with the risk score); MCF is the cubic power of EXTEM-MCF divided by 106; age is in years and not transformed; RF is the reciprocal (1/x) of the respiratory frequency at inclusion.wherep < 0.05;According to this model, the risk of death within 30 days for a patient hospitalized due to COVID-19 was increased with increased age, respiratory frequency, and EXTEM-MCF , as EXTEM-MCF is increased from 65 to 75, mortality risk increases from 0.1 to 0.4% in a 51-year-old patient (lower age quartile), and from 3.8 to 13.7% in a 75-year-old . In this subgroup, we did not observe any significant INTEM-HEPTEM CT difference (p = 0.55) indicating no heparin effect on our ROTEM results.To assess the heparin effect on our test results, we evaluated the difference between INTEM-CT and HEPTEM-CT in all patients receiving LMWH before inclusion .In our dataset, we observed a prolongation of EXTEM-CT, more pronounced in nonsurvivors compared with survivors. A majority of patients in both groups had received LMWH prior to inclusion . Previous in vitro data have shown no effect on EXTEM-CT by LMWH  in therapeutic doses.p = 0.16).The D-dimer-to-P-fibrinogen ratio reflects fibrinolysis in relation to fibrin deposition and has previously been defined as a marker of thrombotic activity, where a higher ratio correlates to a more thrombogenic profile.This study reports a stratification risk score model where we evaluate the ability of ROTEM to predict mortality in COVID-19 patients. Our results support the concept of an early pronounced hypercoagulability, measured by increased EXTEM-MCF on admission that is associated with an increased mortality risk. In combination with age and respiratory frequency, which are two easily measured clinical parameters, our model introduces a feasible tool to assess the risk of death in COVID-19 pneumonia.There is an abundance of findings suggesting that hypercoagulopathy is a crucial component in the pathophysiology of severe COVID-19. Importantly, enhanced anticoagulant treatment has been shown to be associated with reduced mortality,The covariates included in the model  have in earlier studies been suggested as important predictors of clinical outcome. High age is one of the most frequently reported predictors of poor prognosis in COVID-19 and respiratory frequency has in patients with COVID-19 been described as a predictor of mechanical ventilation and in-hospital mortality.In our patients, we typically found prolonged initiation of coagulation (prolonged EXTEM-/INTEM-CT), shortened clot propagation (shortened EXTEM-CFT) with a subsequent forming of a very stable clot with a pronounced clot firmness (increased EXTEM-/FIBTEM-MCF) and coexisting hypofibrinolysis (increased EXTEM-/INTEM-LI60). Our longitudinal analysis supports these findings, where increasing EXTEM-MCF and shortening of EXTEM-CFT were observed confirming enhanced hypercoagulation.Prolonged values of EXTEM-/INTEM-CT are, in contrast to other ROTEM variables, not an indication of hypercoagulation. Increased INTEM-CT in nonsurvivors may be due to anticoagulant treatment effects. Increased EXTEM-CT is in line with previous studies where prolongation of prothrombin time with similar activation proteins as in EXTEM was identified in COVID-19 patients.Low fibrinolytic activity was observed in survivors, as well as nonsurvivors in our data, indicating hypofibrinolysis/fibrinolysis shutdown in both EXTEM and INTEM.Increased levels of P-fibrinogen, corresponding to high FIBTEM-MCF values, indicate pronounced clot stability and are shown to be characteristic for COVID-19 hypercoagulation.We observed elevated levels of P-fibrinogen and D-dimer in our data which is in line with previous studies on COVID-19 and coagulopathy.The D-dimer-to-P-fibrinogen ratio is an indicator of prothrombotic activity where a higher ratio suggests a more pronounced thrombotic state. An increased ratio in COVID-19 may reflect the presence of activated coagulation leading to fibrinogen consumption in the pulmonary vasculature, with simultaneous activation of fibrinolysis resulting in elevated D-dimer levels.Some limitations of this study should be recognized. First, the sample size of 141 patients (of which 18 died within 30 days) was relatively small, given the goal of developing a prediction model. To avoid overfitting, we were limited in the number of predictor variables we evaluated. Second, the data were collected at a single site, and the prediction model was not validated in an independent sample, limiting the generalizability of our findings in other settings and populations. As the data collection was made in the beginning of the SARS-CoV-2 pandemic, no clinical COVID-19 guidelines were yet available, neither were published patient outcome data to compare with, limiting the possibilities of performing external validation at that point. Third, some patients were included somewhat later than the day of admission, which may have reflected test results of different disease stages. Fourth, most patients had received antithrombotic treatment prior to inclusion which may have influenced our laboratory results. However, given that the ROTEM variable we chose to include as a predictor indicates hypercoagulopathy, we do not presume this created any false positive associations.These limitations notwithstanding, we consider our cohort as a representative sample from the first wave of COVID-19 in Stockholm in which a state of hypercoagulability has been shown to be associated with an increased risk of death. Together, these results indicate that ROTEM is a useful analyzing method of coagulopathy in COVID-19 and may be a promising tool to guide anticoagulant treatment.In this study, we evaluated ROTEM as a marker of coagulopathy in COVID-19. First, we presented a risk stratification score model where increased EXTEM-MCF, in combination with age and respiratory frequency, was predictive of increased mortality within 30 days. We then assessed the longitudinal disease course where ROTEM supported the hypothesis of enhanced hypercoagulation in severe disease. Our data did not indicate any effect on EXTEM-CT with increasing LMWH concentrations. In conclusion, ROTEM may be helpful to tailor anticoagulant therapy and suggested as a feasible tool for monitoring disease, which may improve survival in patients with a poor prognosis in COVID-19.Rotational Thromboelastmetry (ROTEM)Extrinsically activated assays with tissue factor (EXTEM)Maximum Clot Firmness (MCF)Activated Partial Thromboplastin Time (APTT)Coagulation Time (CT)Intrinsically activated assays using phospholipid and ellagic acid (INTEM)Corona virus disease 2019 (COVID-19)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)Intensive Care Unit (ICU)Conventional coagulation test (CCT)Polymerase chain reaction (PCR)Computed tomography (CT)Non-invasive ventilation (NIV)Nasal high flow oxygen therapy (NHF)Extracorporeal Membrane Oxygenation (ECMO)Chronic obstructive pulmonary disease (COPD)International Normalized Ratio (INR)Low Molecular Weight Heparin (LMWH)Fibrin-based extrinsically activated assays with tissue factor and platelet inhibitor cytochalasin D (FIBTEM)Intrinsically activated assays with the addition of heparinase (HEPTEM)Clot Formation Time (CFT)Lysis Index (LI)Body mass index (BMI)Area under the curve (AUC)Receiver operating characteristic (ROC)Interquartile range (IQR)Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)Direct oral anticoagulants (DOAC)Respiratory frequency (RF)