Internalization of tissue factor by platelets

Tissue factor (TF) has been found associated with platelets. Mechanisms responsible for TF-platelet interactions and transport are not fully understood. We explored the response of isolated washed platelets to preparations of recombinant (rTF) or placental (pTF) human TF, exposed on lipid microvesicles (MVs). Sequential ultrastructural and immunocytochemical studies revealed trafficking of these TF preparations, being endocytosed by platelets into channels of the open canalicular system (OCS) and accumulating in the cytoplasm and occasionally in alpha-granules. The process of internalization of TF-MVs was accomplished in less than 30 min, being faster for the placental (pTF) than for the recombinant (rTF) preparation. Signs of mild platelet activation with pseudopodia formation were observed at early stages of internalization. Platelets returned to an apparent resting state after 5-10 min. All of these observations paralleled with modifications on patterns of tyrosine phosphorylation for several signaling proteins. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes were accelerated by the presence of other contaminating cellular antigens in the vesicles (pTF). TF carried by platelets could play a potential role in platelet thrombus formation and by extension in the development of ischemic complications.     

Inhibition of tyrosine kinase activity prevents the adhesive and cohesive properties of platelets and the expression of procoagulant activity in response to collagen

Introduction

Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role.

Material and methods

Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation.

Results

Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced.

Conclusions

Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.     

Inhibitory effect of CDP, a polysaccharide extracted from the mushroom Collybia dryophila, on nitric oxide synthase expression and nitric oxide production in macrophages

The effect of Collybia dryophila polysaccharide (CDP), a (1-->3), (1-->4)-beta-D-glucan extracted from the mushroom C. dryophila, was evaluated on nitric oxide (NO) production induced by lipopolysaccharide (LPS) and gamma interferon (IFNgamma) or by LPS alone in RAW 264.7 cells. CDP significantly inhibited NO production in a dose-dependent manner without affecting cell viability. The inhibition of NO by CDP was consistent with decreases in both inducible nitric oxide synthase (iNOS) protein and mRNA expression suggesting that CDP exerts its effect by inhibiting iNOS gene expression. In addition, CDP at concentrations of 400 and 800 microg/ml was shown to significantly increase prostaglandin E2 (PGE2) production in LPS- and IFNgamma-induced macrophages when compared to the control.     

Phosphorylated FTY720 stimulates ERK phosphorylation in astrocytes via S1P receptors

Sphingosine-1-phosphate receptors (S1P1-5) are activated by the endogenous agonist S1P and are expressed in the central nervous system. In astrocytes, activation of S1P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S1P receptors. We examined the effects of FTY720P on ERK phosphorylation and determined which S1P receptor subtype(s) mediated this signaling event. FTY720P augmented ERK phosphorylation in cortical cultures prepared from embryonic day 18 rat brains and was blocked by an MEK inhibitor or by pertussis toxin. Co-localisation of phosphorylated ERK occurred in glial fibrillary acidic protein (GFAP) positive astrocytes but not neurons or oligodendrocytes. Furthermore, FTY720P stimulated ERK phosphorylation in highly enriched astrocyte cultures made from postnatal day 2 rat cortices. The effects of FTY720P were mimicked by selective S1P1 receptor agonists and blocked by S1P1 receptor antagonists. Collectively, these results demonstrate that FTY720P mediates ERK phosphorylation in astrocytes via the activation of S1P1 receptors.     

Elucidating the molecular basis of MSH2‐deficient tumors by combined germline and somatic analysis

In a proportion of patients presenting mismatch repair (MMR)‐deficient tumors, no germline MMR mutations are identified, the so‐called Lynch‐like syndrome (LLS). Recently, MMR‐deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2‐deficient LS‐suspected cases using a comprehensive analysis of colorectal cancer (CRC)‐associated genes at germline and somatic level. Fifty‐eight probands harboring MSH2‐deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty‐five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC‐associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2‐deficient suspected LS tumors.     

Associations between forensic loci and expression levels of neighboring genes may compromise medical privacy

A set of 20 short tandem repeats (STRs) is used by the US criminal justice system to identify suspects and to maintain a database of genetic profiles for individuals who have been previously convicted or arrested. Some of these STRs were identified in the 1990s, with a preference for markers in putative gene deserts to avoid forensic profiles revealing protected medical information. We revisit that assumption, investigating whether forensic genetic profiles reveal information about gene-expression variation or potential medical information. We find six significant correlations (false discovery rate = 0.23) between the forensic STRs and the expression levels of neighboring genes in lymphoblastoid cell lines. We explore possible mechanisms for these associations, showing evidence compatible with forensic STRs causing expression variation or being in linkage disequilibrium with a causal locus in three cases and weaker or potentially spurious associations in the other three cases. Together, these results suggest that forensic genetic loci may reveal expression levels and, perhaps, medical information.

Forensic genetic identification in the United States is typically performed by using genotype data from 20 short tandem repeats (STRs), known as the Combined DNA Index System (CODIS) core loci. Because these markers are highly polymorphic, even just 20 loci provide an immense amount of identifying information regarding a specific individual (1). Thirteen of these CODIS core loci were established by the Federal Bureau of Investigation in 1998. These loci were selected for efficient PCR multiplexing, while maximizing identifying information and minimizing ancestry-based population differences and medically relevant information (2). In 2017, seven additional STRs were added to the CODIS core loci, selected for similar criteria, particularly no known associations with medical conditions (3).It is important from a legal standpoint that CODIS genotypes do not reveal medical information. Laws authorizing the compulsory collection of DNA from certain persons may come into conflict with state privacy statutes or the US Constitution if medical information is embedded (4). In fact, hundreds of court cases rely on the premise that the CODIS variants are uninformative, often citing this quote relating to the DNA Analysis Backlog Elimination Act of 2000, which states that the CODIS loci “were purposely selected because they are not associated with any known physical or medical characteristics” (Letter from Robert Raben, Assistant Attorney General, to Judiciary Committee Chairman Henry Hyde) (5). Yet, some of the CODIS loci, particularly those selected before the human genome was sequenced, are very close to genes. In fact, 11 of the CODIS loci are intronic (6).Any trait information conveyed by CODIS genotypes would raise questions regarding the medical privacy of individuals whose CODIS profiles are compelled by the government, as well as their genetic relatives. These include over 19,350,445 people arrested or convicted whose CODIS profiles are held in the US national database (7), a group that overrepresents people of color, especially Black populations (8). The historical and current treatment of arrested and convicted individuals is rife with rights unjustly curtailed, raising even more concern about a potentially lax approach to medical privacy for this population (9–11). While the access-limited, federally regulated national database is vast, it does not include all CODIS profiles held at state and local levels. For instance, in the “spit and acquit” practice by the Orange County (CA) District Attorney’s Office, certain misdemeanor defendants can be offered a dismissal in exchange for a DNA sample, resulting in a local database of over 150,000 individuals (12). Other expansive local practices include using samples collected from nonsuspects for criminal investigation. A recent example is a sexual assault survivor’s DNA profile later being used to connect her to a property crime (13, 14). Given the scope of individuals with CODIS profiles stored and/or shared, in this study, we re-examine the assumption that CODIS genotypes have no functional or medical impact.It has long been known that variation in STR number can alter gene function and regulation, sometimes resulting in dramatic phenotypes. A classic example is the coding STR expansion in the HD gene, which causes increasingly severe Huntington’s disease (15). Noncoding STRs have also been found to impact gene expression, resulting in trait variation. For instance, large numbers of repeats in an STR in the 5′ untranslated region of FRAXA impacts local methylation and gene regulation, causing Fragile X syndrome (15).More recent studies involving genome-wide surveys have found thousands of replicable associations between STR length and gene expression level (16–18). STR length variation can impact methylation, as well as histone modifications, causing evolutionarily conserved changes in gene expression (16, 17). Some of these STR-associated expression changes were associated with clinical traits (16). Somatic STR mutations have been implicated in the development of cancer (19). One recent analysis showed that individuals with autism have significantly more de novo STR mutations (particularly in introns), as compared with their neurotypical siblings (20). This growing body of evidence suggests that STR length variation is causally responsible for a range of complex trait variations, including pathogenic conditions (21).These results raise questions about whether the CODIS loci could impact medically relevant traits. Based on data available in 2011, a review of phenotypic associations with genetic loci concluded that there were no significant associations with the CODIS STRs (6). However, the study did report that some CODIS loci fall within predicted sites for genomic regulation, and all CODIS loci are within 1 kb of at least one genetic variant associated with a phenotype (6). Because the linkage disequilibrium (LD) surrounding the CODIS loci is strong enough to infer the genotypes of surrounding single-nucleotide polymorphisms (SNPs) (21–,23), phenotype information may be inferable through the CODIS genotypes. A more recent review of literature has identified 84 significant published associations between traits and STRs for 18 of the 20 CODIS loci (25).Here, we investigate whether genotypes at the CODIS loci could directly reveal information about a fundamental trait: the expression levels of neighboring genes. We identify CODIS loci significantly correlated with the expression of nearby genes (CODISeSTRs). We shed light on the mechanisms underlying these associations. First, we consider the possibility that the associations are caused by population structure as a confounding factor by testing for expression–genotype associations within subpopulations. With population stratification ruled out, we explore the possibility of CODISeSTRs causing expression variation by both comparing their genomic features to a panel of STRs with strong evidence of expression impact (18) and using a fine-mapping framework (CAVIAR) to identify putative causal loci (26). Finally, we investigate the hypothesis that CODISeSTRs may be in LD with a causal variant by examining their LD with putative causal sites identified by CAVIAR, as well as DNase I hypersensitivity (DHS) sites.     

Association between severe hypoglycaemia and risk of dementia in patients with type 2 diabetes mellitus: A systematic review and meta-analysis

The aim of this systematic review was to analyse whether there is an association between severe hypoglycaemia and the incidence of dementia in patients with type 2 diabetes mellitus. We systematically searched the MEDLINE, Scopus, and Cochrane databases from their inception until September 2022 for observational studies on the association between hypoglycaemia and the risk of dementia. The DerSimonian and Laird method was used to compute a pooled estimate of the risk for such association. Risk ratio (RR) and its respective 95% confidence interval (95% CI). Two analyses were performed to estimate the risk of dementia: (i) any hypoglycaemia versus no hypoglycaemia and (ii) a dose–response analysis for one, two, or more than three hypoglycemic events versus no hypoglycaemia. PROSPERO registration number CRD42020219200. Seven studies were included. The pooled RR for the association of severe hypoglycaemia and risk of dementia was 1.47 (95% CI: 1.24–1.74). When the dose–response trend was analysed, the pooled RR for the risk of dementia was increased according to the hypoglycaemia events as follows: 1.29 (95% CI: 1.15–1.44) for one hypoglycemic event; 1.68 (95% CI: 1.38–2.04) for two hypoglycemic events; and 1.99 (95% CI: 1.48–2.68) for three or more hypoglycemic events. Our study demonstrates a 54% higher risk of dementia among people who suffer a hypoglycaemia event compared to nonhypoglycaemia. Considering our results and the prevalence of people suffering from diabetes mellitus, health education for both newly diagnosed and already diagnosed people could be a useful tool for glycaemic control, thus avoiding hypoglycaemic events.