Psychosocial impact of the COVID-19 pandemic on 4378 UK healthcare workers and ancillary staff: initial baseline data from a cohort study collected during the first wave of the pandemic.

OBJECTIVES: This study reports preliminary findings on the prevalence of, and factors associated with, mental health and well-being outcomes of healthcare workers during the early months (April-June) of the COVID-19 pandemic in the UK. METHODS: Preliminary cross-sectional data were analysed from a cohort study (n=4378). Clinical and non-clinical staff of three London-based NHS Trusts, including acute and mental health Trusts, took part in an online baseline survey. The primary outcome measure used is the presence of probable common mental disorders (CMDs), measured by the General Health Questionnaire. Secondary outcomes are probable anxiety (seven-item Generalised Anxiety Disorder), depression (nine-item Patient Health Questionnaire), post-traumatic stress disorder (PTSD) (six-item Post-Traumatic Stress Disorder checklist), suicidal ideation (Clinical Interview Schedule) and alcohol use (Alcohol Use Disorder Identification Test). Moral injury is measured using the Moray Injury Event Scale. RESULTS: Analyses showed substantial levels of probable CMDs (58.9%, 95% CI 58.1 to 60.8) and of PTSD (30.2%, 95% CI 28.1 to 32.5) with lower levels of depression (27.3%, 95% CI 25.3 to 29.4), anxiety (23.2%, 95% CI 21.3 to 25.3) and alcohol misuse (10.5%, 95% CI 9.2 to 11.9). Women, younger staff and nurses tended to have poorer outcomes than other staff, except for alcohol misuse. Higher reported exposure to moral injury (distress resulting from violation of one's moral code) was strongly associated with increased levels of probable CMDs, anxiety, depression, PTSD symptoms and alcohol misuse. CONCLUSIONS: Our findings suggest that mental health support for healthcare workers should consider those demographics and occupations at highest risk. Rigorous longitudinal data are needed in order to respond to the potential long-term mental health impacts of the pandemic.

Comparative Platelet Releasate Proteomic Profiling of Acute Coronary Syndrome versus Stable Coronary Artery Disease.

Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. Here, we undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1 U/ml thrombin-induced PR from 13 acute coronary syndrome vs. 14 stable angina pectoris patients using a tandem mass spectrometry approach. Data are available via ProteomeXchange with identifier PXD009356. 318 PR proteins were identified across both cohorts with 9 proteins found to be differentially released, including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5), and fibronectin (FN1). Strikingly, these 9 differential proteins were all associated with the gene ontology cellular component term "extracellular vesicle" and reduced levels of EVs were detected in the corresponding plasma of ST-segment elevation myocardial infarction (STEMI) patients. Network analysis revealed 3 proteins either reduced (F5; FN1) or absent (CLEC3B) in the PR of STEMI patients that are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated proteomic signature may prove useful for non-invasive risk assessment of the progression of coronary artery disease. These data further contribute to the growing evidence-base of using the platelet releasate as a predictor of pathological state and disease severity.

The Platelet Releasate is Altered in Human Pregnancy.

PURPOSE: Healthy pregnancy is characterized by an increase in platelet activation and a decrease in the number of circulating platelets with gestation. Despite this recognized importance, proteomic studies investigating platelets in healthy pregnancy have not been performed. As platelet cargo can be altered in different conditions, it is hypothesized that platelets may store a relevant and bespoke collection of molecules during pregnancy. EXPERIMENTAL DESIGN: Comparative label-free quantitative proteomic profiling of platelet releasates (PRs) is performed from 18 healthy pregnant and 13 non-pregnant women using an MS/MS approach. RESULTS: Of the 723 proteins identified, 69 PR proteins are found to be differentially released from platelets in pregnancy, including proteins only expressed during pregnancy such as pregnancy-specific glycoproteins and human placental lactogen. Moreover, the population of exosomal vesicles present in the PR is also modified in pregnancy. Receiver operating characteristic analysis shows the predictive ability of 11 PR proteins to distinctly classify pregnant and nonpregnant women with an area under the curve of 0.876, a sensitivity of 88.9%, and a specificity of 84.6%. CONCLUSIONS AND CLINICAL RELEVANCE: Taken together this demonstrates that platelets and their released cargo are 'educated' in physiologic stressful conditions such as pregnancy and may represent a promising platform to study pregnancy complications.

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Proteomic Analysis Reveals a Strong Association of ß-Catenin With Cadherin Adherens Junctions in Resting Human Platelets.

It was previously demonstrated that the WNT/ß-catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT-3a, suppresses platelet adhesion and activation. In nucleated cells, ß-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell-cell adhesion. The specific role of ß-catenin in the anucleate platelet however remains elusive. Here, a label-free quantitative proteomic analysis of ß-catenin immunoprecipitates from human platelets is performed and nine co-immunoprecipitating proteins are identified. Three of the co-immunoprecipitating proteins (α-catenin-1, cadherin-6, and ß-catenin-interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet ß-catenin and cadherin-6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for ß-catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins.

A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids.

Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.

Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype.

Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbß3, while binding also occurred in a CX3CL1-CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbß3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.

Effect of platelet-derived ß-thromboglobulins on coagulation.

BACKGROUND: ß-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived ß-thromboglobulins (ßTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of ßTG on coagulation is unknown. AIM/METHODS: Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified ßTG on coagulation. RESULTS: In normal pooled plasma, ßTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, ßTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when ßTG was incubated with factor X, suggesting a direct interaction between ßTG and factor X. Using SPR, ßTG were found to bind to immobilised factor X in a dose dependent manner. CONCLUSION: ßTG modulate coagulation in vitro via an interaction with factor X.

Thrombin generation correlates with disease duration in multiple sclerosis (MS): Novel insights into the MS-associated prothrombotic state.

BACKGROUND: Thrombin is well recognised for its role in the coagulation cascade but it also plays a role in inflammation, with enhanced thrombin generation observed in several inflammatory disorders. Although patients with multiple sclerosis (MS) have a higher incidence of thrombotic disease, thrombin generation has not been studied to date. OBJECTIVES: The aim of this study was to characterise calibrated automated thrombography parameters in patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS) in comparison to healthy controls (HCs). METHODS: Calibrated automated thrombography was performed on platelet poor plasma from 15 patients with RRMS, 15 with PPMS and 19 HCs. RESULTS: We found that patients with RRMS generate thrombin at a significantly faster rate than the less inflammatory subtype, PPMS or HCs. In addition, the speed of thrombin generation was significantly correlated with time from clinical diagnosis in both subtypes. However, in RRMS the rate of thrombin generation was increased with increased time from clinical diagnosis, while in PPMS the rate of thrombin generation decreased with increased time from clinical diagnosis. CONCLUSIONS: These data likely reflect the differential active proinflammatory states in each MS subtype and provide novel mechanistic insights into the clinically relevant prothrombotic state observed in these patients.

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3ß (Ser9), nuclear accumulation of ß-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-ß (TGF-ß); Wnt6 reversed TGF-ß-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-ß-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/ß(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.

The reactive chemisorption of alkyl iodides at Cu(110) and Ag(111) surfaces: a combined STM and XPS study.

The chemisorption of methyl and phenyl iodide has been studied at Cu(110) and Ag(111) surfaces at 290 K with STM and XPS. At both surfaces dissociative adsorption of both molecules leads to chemisorbed iodine, with the STM showing c(2 x 2) and (square root 3 x square root 3)R30 structures at the Cu(110) and Ag(111) surfaces, respectively. At the Cu(110) surface a comparison of coexisting c(2 x 2) I(a) and p(2 x 1) O(a) domains shows the iodine adatoms to be chemisorbed in hollow sites with evidence at low coverage for diffusion in the (110) direction. In the case of methyl iodide no carbon adsorption is observed at either the silver or the copper surfaces, but chemisorbed phenyl groups are imaged at the Cu(110) surface after exposure to phenyl iodide. The STM images show the phenyl groups as bright features approximately 0.7 nm in diameter and 0.11 nm above the iodine adlayer, reaching a maximum surface concentration after approximately 6 Langmuir exposure. However, the phenyl coverage decreases with subsequent exposures to PhI and is negligible by approximately 1000 L exposure, consistent with the formation and desorption of biphenyl. The adsorbed phenyls are located above hollow sites in the substrate, they are stabilized at the top and bottom of step edges and in paired chains (1.1 nm apart) on the terraces with a regular interphenyl spacing within the chains of 1.0 nm in the (110) direction. The interphenyl ring spacing and diffusion of individual phenyls from within the chains shows that the chains do not consist of biphenyl species but may be a precursor to their formation. Although the XPS data shows carbon present at the Ag(111) surface after exposure to PhI, no features attributable to phenyl groups were observed by STM.