Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked

Does Gender or Religion Contribute to the Risk of COVID-19 in Hospital Doctors?

This webpage details and provides the research study conducted in the United Kingdom through online surveys focusing on the relationship between healthcare workplace prevention efforts, COVID-19 risks, religious identity, and gender. The research study focuses on healthcare workers, primarily hospital doctors and mental health doctors. A PDF of the entire study is available on the webpage

Impact of Scotland’s comprehensive, smoke-free legislation on stroke

&lt;p&gt;Background: Previous studies have reported a reduction in acute coronary events following smoke-free legislation. Evidence is lacking on whether stroke is also reduced. The aim was to determine whether the incidence of stroke, overalland by sub-type, fell following introduction of smoke-free legislation across Scotland on 26 March 2006.&lt;/p&gt; &lt;p&gt;Methods and Findings: A negative binomial regression model was used to determine whether the introduction of smokefree legislation resulted in a step and/or slope change in stroke incidence. The model was adjusted for age-group, sex, socioeconomic deprivation quintile, urban/rural residence and month. Interaction tests were also performed. Routine hospital administrative data and death certificates were used to identify all hospital admissions and pre-hospital deaths due to stroke (ICD10 codes I61, I63 and I64) in Scotland between 2000 and 2010 inclusive. Prior to the legislation, rates of all stroke, intracerebral haemorrhage and unspecified stroke were decreasing, whilst cerebral infarction was increasing at 0.97% per annum. Following the legislation, there was a dramatic fall in cerebral infarctions that persisted for around 20 months. No visible effect was observed for other types of stroke. The model confirmed an 8.90% (95% CI 4.85, 12.77, p,0.001) stepwise reduction in cerebral infarction at the time the legislation was implemented, after adjustment for potential cofounders.&lt;/p&gt; &lt;p&gt;Conclusions: Following introduction of national, comprehensive smoke-free legislation there was a selective reduction in cerebral infarction that was not apparent in other types of stroke.&lt;/p&gt

Circulating extracellular vesicles induce monocyte dysfunction and are associated with sepsis and high mortality in cirrhosis

BACKGROUND: Sepsis is common in cirrhosis and is often a result of immune dysregulation. Specific stimuli and pathways of inter-cellular communications between immune cells in cirrhosis and sepsis are incompletely understood. Immune cell-derived Extracellular Vesicles (EV) were studied to understand mechanisms of sepsis in cirrhosis. METHODS: Immune-cell derived EV were measured in cirrhosis patients [Child-Turcotte-Pugh (Child) score A, n=15; B n=16; C n=43 and Child-C with sepsis (n=38)], and healthy controls (HC, n=11). In-vitro and in-vivo functional relevance of EV in cirrhosis and associated sepsis was investigated. RESULTS: Monocyte, neutrophil and hematopoietic stem cells associated EV progressively increased with higher Child score (p0.3, p<0.001), which further increased in Child C sepsis than without sepsis(p<0.001); monocyte EV showing the highest association with disease stage [p=0.013; Odds ratio-4.14(1.34-12.42)]. A threshold level of monocyte EV of 53/µl predicted mortality in patients of Child C with sepsis [Odds ratio-6.2 (2.4-15.9), AUROC=0.76, p<0.01]. In vitro EV from cirrhotic with sepsis compared without sepsis, induced mobilization arrest in healthy monocytes within 4 hours (p=0.004), reduced basal oxygen consumption rate (p<0.001) and induced pro-inflammatory genes (p<0.05). The septic-EV on adoptive transfer to C57/BL6J mice, induced sepsis like condition within 24h with leukocytopenia (p=0.005), intrahepatic inflammation with increased CD11b+ cells (p=0.03) and bone marrow hyperplasia (p<0.01). CONCLUSION: Extracellular vesicles induce functional impairment in circulating monocytes and contribute to the development and perpetuation of sepsis. High levels of monocyte EV correlate with mortality and can help early stratification of sicker patients

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients

Co3O4 Nanocrystals on Graphene as a Synergistic Catalyst for Oxygen Reduction Reaction

Catalysts for oxygen reduction and evolution reactions are at the heart of key renewable energy technologies including fuel cells and water splitting. Despite tremendous efforts, developing oxygen electrode catalysts with high activity at low costs remains a grand challenge. Here, we report a hybrid material of Co3O4 nanocrystals grown on reduced graphene oxide (GO) as a high-performance bi-functional catalyst for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). While Co3O4 or graphene oxide alone has little catalytic activity, their hybrid exhibits an unexpected, surprisingly high ORR activity that is further enhanced by nitrogen-doping of graphene. The Co3O4/N-doped graphene hybrid exhibits similar catalytic activity but superior stability to Pt in alkaline solutions. The same hybrid is also highly active for OER, making it a high performance non-precious metal based bi-catalyst for both ORR and OER. The unusual catalytic activity arises from synergetic chemical coupling effects between Co3O4 and graphene.Comment: published in Nature Material

Uteroplacental adenovirus VEGF gene therapy increases fetal growth velocity in growth-restricted sheep pregnancies.

Introduction: Fetal growth restriction (FGR) occurs in ~8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterised by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad) mediated over-expression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from mid-gestation correlates with reduced lamb birthweight near term. Materials and Methods: Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high-intake (n=45) or control-intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomised mid-gestation to receive bilateral UtA injections of 5x1011 particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14) or control saline (n=13). Fetal growth/wellbeing were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularisation within the perivascular adventitia and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry and qRT-PCR, respectively. Results: Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3-4 weeks post-injection (p=0.016-0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) following Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter to abdominal circumference and brain to liver weight ratios). No effects were observed on UBF or neovascularisation, however Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal to placental weight ratio) and FLT1/KDR mRNA expression was increased in the maternal but not fetal placental compartments, suggesting downstream effects on placental function. Conclusion: Ad.VEGF gene therapy improves fetal growth in a sheep model of FGR, although the precise mechanism of action remains unclear

The intersection of COVID-19 and autoimmunity

Acute coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is characterized by diverse clinical presentations, ranging from asymptomatic infection to fatal respiratory failure, and often associated with varied longer-term sequelae. Over the past 18 months, it has become apparent that inappropriate immune responses contribute to the pathogenesis of severe COVID-19. Researchers working at the intersection of COVID-19 and autoimmunity recently gathered at an American Autoimmune Related Disease Association (AARDA) Noel R. Rose Colloquium to address the current state of knowledge regarding two important questions: Does established autoimmunity predispose to severe COVID-19? And, at the same time, can SARS-CoV-2 infection trigger de novo autoimmunity? Indeed, work to date has demonstrated that 10 to 15% of patients with critical COVID-19 pneumonia exhibit autoantibodies against type I interferons, suggesting that preexisting autoimmunity underlies severe disease in some patients. Other studies have identified functional autoantibodies following infection with SARS-CoV-2, such as those that promote thrombosis or antagonize cytokine signaling. These autoantibodies may arise from a predominantly extrafollicular B cell response that is more prone to generating autoantibody-secreting B cells. This review highlights the current understanding, evolving concepts, and unanswered questions provided by this unique opportunity to determine mechanisms by which a viral infection can be exacerbated by, and even trigger, autoimmunity. The potential role of autoimmunity in post-acute sequelae of COVID-19 is also discussed

Prognostic impact of human epidermal growth factor-like receptor 2 and hormone receptor status in inflammatory breast cancer (IBC): analysis of 2,014 IBC patient cases from the California Cancer Registry

IntroductionInflammatory breast cancer (IBC) is an aggressive form of breast cancer associated with overexpression of Her2/Neu (human epidermal growth factor-like receptor 2 (HER2)) and poor survival. We investigated survival differences for IBC patient cases based on hormone receptor status and HER2 receptor status using data from the California Cancer Registry, as contrasted with locally advanced breast cancer (LABC), metastatic breast cancer (MBC) and non-T4 breast cancer.MethodsA case-only analysis of 80,099 incident female breast cancer patient cases in the California Cancer Registry during 1999 to 2003 was performed, with follow-up through March 2007. Overall survival (OS) and breast cancer-specific survival (BC-SS) were analyzed using Kaplan-Meier methods and Cox proportional hazards ratios.ResultsA total of 2,014 IBC, 1,268 LABC, 3,059 MBC, and 73,758 non-T4 breast cancer patient cases were identified. HER2+ was associated with advanced tumor stage (P &lt; 0.0001). IBC patient cases were more likely to be HER2+ (40%) and less likely to be hormone receptor-positive (HmR+) (59%) compared with LABC (35% and 69%, respectively), MBC (35% and 74%), and non-T4 patient cases (22% and 82%). HmR+ status was associated with improved OS and BC-SS for each breast cancer subtype after adjustment for clinically relevant factors. In multivariate analysis, HER2+ (versus HER2-) status was associated with poor BC-SS for non-T4 patient cases (hazards ratio = 1.16, 95% confidence interval 1.05 to 1.28) and had a borderline significant association with improved BC-SS for IBC (hazards ratio = 0.82, 95% confidence interval = 0.68 to 0.99).ConclusionsDespite an association with advanced tumor stage, HER2+ status is not an independent adverse prognostic factor for survival among IBC patient cases