Background An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19. Methods In this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan-Meier analysis was used to further explore the association between biochemical markers and survival. Findings 68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0•0001) and soluble P-selectin (15•9 ng/mL [4•8] vs 11•2 ng/mL [3•1]; p=0•0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r=0•38; p=0•0022) and soluble thrombomodulin (r=0•38; p=0•0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3•26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0•0050) and lower likelihood of survival on Kaplan-Meier analysis (hazard ratio 5•9, 95% CI 1•9-18•4; p=0•0087).Interpretation Our findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19.

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers: Washington University School of Medicine (n=134) and Yale School of Medicine (n=49). We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza (n=54), and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV, n=22). We demonstrate that circulating markers of complement activation are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., angiopoietin-2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T 3) produced 411 mg͞liter scopolamine, which was over nine times more than that in the wild type (43 mg͞liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184 mg͞liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine N-methyltransferase ͉ scopolamine ͉ transformation

This paper introduces a method for feature extraction and emotion recognition based on empirical mode decomposition (EMD). By using EMD, EEG signals are decomposed into Intrinsic Mode Functions (IMFs) automatically. Multidimensional information of IMF is utilized as features, the first difference of time series, the first difference of phase, and the normalized energy. The performance of the proposed method is verified on a publicly available emotional database. The results show that the three features are effective for emotion recognition. The role of each IMF is inquired and we find that high frequency component IMF1 has significant effect on different emotional states detection. The informative electrodes based on EMD strategy are analyzed. In addition, the classification accuracy of the proposed method is compared with several classical techniques, including fractal dimension (FD), sample entropy, differential entropy, and discrete wavelet transform (DWT). Experiment results on DEAP datasets demonstrate that our method can improve emotion recognition performance.

Despite over 30 million infected and 800,000 deaths, the pathophysiological factors that determine the wide spectrum of clinical outcomes in COVID-19 remain inadequately defined. Importantly, patients with underlying cardiovascular disease have been found to have worse clinical outcomes (1), and autopsy findings of endotheliopathy in COVID-19 have accumulated (2). Nonetheless, circulating markers of endothelial or vascular injury associated with disease severity and mortality have not been reliably established. To address this limitation and better understand COVID-19 pathogenesis, we report plasma profiling of factors related to the vascular system from patients admitted to our Hospital with confirmed COVID-19 diagnosis, which revealed significant increases in markers of angiogenesis and endotheliopathy in hospitalized patients.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

Insufficient lysosomal removal of autophagic cargoes in cardiomyocytes has been suggested as a main cause for the impairment of the autophagic-lysosomal pathway (ALP) in many forms of heart disease including cardiac proteinopathy and may play an important pathogenic role; however, the molecular basis and the correcting strategy for the cardiac ALP insufficiency require further investigation. The present study was sought to determine whether myocardial expression and activity of TFEB, the recently identified ALP master regulator, are impaired in a cardiac proteinopathy mouse model and to determine the effect of genetic manipulation of TFEB expression on autophagy and proteotoxicity in a cardiomyocyte model of proteinopathy. We found that increased myocardial TFEB mRNA levels and a TFEB protein isoform switch were associated with marked decreases in the mRNA levels of representative TFEB target genes and increased mTORC1 activation, in mice with cardiac transgenic expression of a missense (R120G) mutant αB-crystallin (CryABR120G), a well-established model of cardiac proteinopathy. Using neonatal rat ventricular cardiomyocyte cultures, we demonstrated that downregulation of TFEB decreased autophagic flux in cardiomyocytes both at baseline and during CryABR120G overexpression and increased CryABR120G protein aggregates. Conversely, forced TFEB overexpression increased autophagic flux and remarkably attenuated the CryABR120G overexpression- induced accumulation of ubiquitinated proteins, caspase 3 cleavage, LDH leakage, and decreases in cell viability. Moreover, these protective effects of TFEB were dramatically diminished by inhibiting autophagy. We conclude that myocardial TFEB signaling is impaired in cardiac proteinopathy and forced TFEB overexpression protects against proteotoxicity in cardiomyocytes through improving ALP activity.