Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odds ratio (95%CI) 4.1 (3.3-4.8), 3.9 (2.6-5.1) and 3.6 (2.0-5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5 (0.9-2.1)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay.

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3’-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40–56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

SummaryWe here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

SummaryMolecular diagnosis of von Willebrand Disease (VWD) is particularly complex. The autosomal von Willebrand factor gene (VWF) is large and highly polymorphic, and there is a highly homologous (>96%) partial pseudogene in chromosome 22. Because of these difficulties, application of molecular study of VWD to the clinical routine has been considerably delayed. Recent advances in sequencing technology and bioinformatics could convert direct sequencing of the complete VWF into a routine diagnostic tool for VWD, which is especially desirable in types 1 and 3. This study describes a highly optimized procedure in which all the coding and intronic flanking regions of VWF are amplified under identical thermocycling parameters in a ready-to-use PCR plate format. The entire sequencing procedure, from blood extraction to mutation identification, can be done within 24 hours, resulting in a simple, versatile, cost-effective strategy with little hands-on time requirements. To validate the method, we performed full-length VWF sequencing of 21 index cases including seven of each VWD type. A total of 30 VWF genetic variations were identified. Twelve of these sequence variations are new, including four missense, one nonsense, one insertion, the first insertion-deletion described in VWF, and 5 potential splice site mutations. To our knowledge, this is the fastest and most efficient protocol designed to date for complete sequencing of the VWF coding region in the molecular diagnosis of VWD.

SARS-CoV-2 has been associated with an increased rate of venous thromboembolism in critically ill patients. Since surgical patients are already at higher risk of venous thromboembolism than general populations, this study aimed to determine if patients with peri-operative or prior SARS-CoV-2 were at further increased risk of venous thromboembolism. We conducted a planned sub-study and analysis from an international, multicentre, prospective cohort study of elective and emergency patients undergoing surgery during October 2020. Patients from all surgical specialties were included. The primary outcome measure was venous thromboembolism (pulmonary embolism or deep vein thrombosis) within 30 days of surgery. SARS-CoV-2 diagnosis was defined as peri-operative (7 days before to 30 days after surgery); recent (1-6 weeks before surgery); previous (≥7 weeks before surgery); or none. Information on prophylaxis regimens or pre-operative anti-coagulation for baseline comorbidities was not available. Postoperative venous thromboembolism rate was 0.5% (666/123,591) in patients without SARS-CoV-2; 2.2% (50/2317) in patients with peri-operative SARS-CoV-2; 1.6% (15/953) in patients with recent SARS-CoV-2; and 1.0% (11/1148) in patients with previous SARS-CoV-2. After adjustment for confounding factors, patients with peri-operative (adjusted odds ratio 1.5 (95%CI 1.1-2.0)) and recent SARS-CoV-2 (1.9 (95%CI 1.2-3.3)) remained at higher risk of venous thromboembolism, with a borderline finding in previous SARS-CoV-2 (1.7 (95%CI 0.9-3.0)). Overall, venous thromboembolism was independently associated with 30-day mortality ). In patients with SARS-CoV-2, mortality without venous thromboembolism was 7.4% (319/4342) and with venous thromboembolism was 40.8% (31/76). Patients undergoing surgery with peri-operative or recent SARS-CoV-2 appear to be at increased risk of postoperative venous thromboembolism compared with patients with no history of SARS-CoV-2 infection. Optimal venous thromboembolism prophylaxis and treatment are unknown in this cohort of patients, and these data should be interpreted accordingly.

The nitric oxide synthase family of proteins is the unique class of mammalian enzymes that metabolizes L-arginine to form nitric oxide (NO). The atherogenic action of low-density lipoproteins (LDL) may be mediated, in part, by its effects on endothelial-derived nitric oxide. To determine whether native LDL (nLDL), at atherogenic concentrations, are capable of modulating NO synthase expression, we treated human umbilical vein endothelial cells with increasing concentrations of human nLDL (0Ϫ240 mg cholesterol/dl) for various time periods (2Ϫ48 h). Northern and western blot analyses indicate that both endothelial NO-synthase mRNA and protein are down-regulated by atherogenic concentrations of nLDL (180 and 240 mg cholesterol/dl) after 48 h of incubation. Cycloheximide and actinomycin D experiments suggest that this down-regulation operates at a transcriptional level. Additionally, treatment of the cells with high-density lipoproteins, at human physiological concentrations (45 mg cholesterol/dl), does not appear to alter the expression of endothelial NO synthase which seems to indicate that nLDL affect the gene transcription rate by a specific and concentration-dependent mechanism. These findings may have important implications because they provide a novel mechanism by which hypercholesterolemia induces early changes on endothelial cells that could have pathophysiological significance in the atherosclerotic process.Keywords : endothelial cells; human umbilical vein endothelial cells; hypercholesterolemia ; low-density lipoprotein; nitric oxide.The endothelium regulates vascular tone by producing pros-On the other hand, hypercholesterolemia is generally associated with an increase in low-density lipoprotein (LDL), which are the tacyclin, nitric oxide (NO) and endothelin, among other vasoactive compounds, in response to several stimuli [1]. Alterations major carriers of cholesterol in the blood. High LDL levels are a known risk factor for the development of atherosclerosis but in endothelial function and related processes are known to be involved in the pathogenesis of many cardiovascular diseases, the mechanisms by which LDL acts and if there is a relationship with NOS regulation is uncertain. It has been reported that LDL including hypertension and atherosclerosis [2].NO, a potent vasodilator, is also released by other cell types: enhance prostacyclin production [6], endocytic activity [7], permeability [8], recruitment of mononuclear cells [9] and synthesis smooth muscle cells, neurons, macrophages and hepatocytes. NO is generated by conversion of L-arginine to L-citrulline by of adhesion molecules [10] in endothelial cells. These findings are important since some of them participate in the atherogenic enzymatic action of NO synthases (NOS). Three isoforms of process and can be inhibited by NO. However, the role of NO NOS have been cloned and characterized: two Ca 2ϩ /calmodulinon endothelial dysfunction associated with hypercholesterolemia dependent isoforms, constitutively expressed in brain and endoor atheroscl...