Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy

Background Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma. Results We compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups. Conclusion The miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression

Prognostic utility of ADAMTS13 activity for the atypical hemolytic uremic syndrome (aHUS) and comparison of complement serology between aHUS and thrombotic thrombocytopenic purpura

Background Atypical hemolytic uremic syndrome (aHUS) involves dysregulation of the complement system, but whether this also occurs in thrombotic thrombocytopenic purpura (TTP) remains unclear. Although these conditions are difficult to differentiate clinically, TTP can be distinguished by low (<10%) ADAMTS13 activity. The aim was to identify the differences in complement activation products between TTP and aHUS and investigate ADAMTS13 activity as a prognostic factor in aHUS. Methods We analyzed patients with thrombotic microangiopathy diagnosed as TTP (N=48) or aHUS (N=50), selected from a Korean registry (N=551). Complement activation products in the plasma samples collected from the patients prior to treatment and in 40 healthy controls were measured by ELISA. Results The levels of generalized (C3a), alternate (factor Bb), and terminal (C5a and C5b-9) markers were significantly higher (all P<0.01) in the patients than in the healthy controls. Only the factor Bb levels significantly differed (P=0.008) between the two disease groups. In aHUS patients, high normal ADAMTS13 activity (≥77%) was associated with improved treatment response (OR, 6.769; 95% CI, 1.605–28.542; P=0.005), remission (OR, 6.000; 95% CI, 1.693–21.262; P=0.004), exacerbation (OR, 0.242; 95% CI, 0.064–0.916; P=0.031), and disease-associated mortality rates (OR, 0.155; 95% CI, 0.029–0.813; P=0.017). Conclusion These data suggest that complement biomarkers, except factor Bb, are similarly activated in TTP and aHUS patients, and ADAMTS13 activity can predict the treatment response and outcome in aHUS patients

Effect of dialysate composition on intercompartmental fluid shift

Effect of dialysate composition on intercompartmental fluid shift. Effect of dialysate composition on intercompartmental fluid shift and hemodynamics was studied in 12 patients during 1.5 or 2 hours of hemodialysis without net ultrafiltration, using high (H;Na 154 mmol/liter), normal (N;Na 140 mmol/liter) or low (L:Na 126 mmol/liter) concentration dialysate. H dialysate was associated with a small (0.9%) increase in blood volume, a larger increase in plasma volume and a decrease in erythrocyte volume. L dialysate resulted in a 2.3% decrease in blood volume, a larger decrease in plasma volume and an increase in erythrocyte volume. N dialysate gave results which were intermediately between the other two dialysis conditions. There was no difference in the post-dialysis mean arterial pressure between the groups, although heart rate increased more during H dialysis than during the other two conditions. Change in blood and erythrocyte volume correlated significantly with change in plasma Na concentration and osmolality, but not with change in plasma urea concentration. We conclude that dialysate composition affects the movement of water into and out of the plasma and erythrocytes in a manner that can be accounted for by altered plasma concentrations of osmotically active substances

Dimeric FcγR ectodomains detect pathogenic anti-platelet factor 4-heparin antibodies in heparin-induced thromobocytopenia

Background Heparin‐induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)–heparin complexes following heparin exposure. Not all anti‐PF4–heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis. Objectives To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/Methods Plasma from 27 suspected HIT patients were tested for pathogenic anti‐PF4–heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4–heparin IgG ELISA, the HemosIL AcuStar HIT IgG‐specific assay, and a serotonin release assay (SRA). Results The dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4–heparin immune complexes. Conclusions This study found the cell‐free, function‐based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG‐specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts

1,25(OH)2D3 administration in moderate renal failure: A prospective double-blind trial

1,25(OH)2D3 administration in moderate renal failure: A prospective double blind trial. This study represents the first randomized prospective, double-blind, placebo-controlled trial of the efficacy of 1,25(OH)2D3 on bone histology and serum biochemistry in patients with mild to moderate renal failure. Sixteen patients with chronic renal impairment (creatinine clearance 20 to 59ml per min) received either 1,25(OH)2D3, at a dose of 0.25 to 0.5 µg daily (eight patients), or placebo. Transiliac crest bone biopsies were performed before entrance into the study and after 12 months of experimental observation. None of the patients were symptomatic or had radiological evidence of bone disease. Of the thirteen patients who completed the study, initial serum 1,25(OH)2D levels were low in seven patients and parathyroid hormone levels were elevated in seven patients. Bone histology was abnormal in all patients. 1,25(OH)2D3 treatment was associated with a significant fall in serum phosphorus and alkaline phosphatase concentrations as well as with histological evidence of an amelioration of hyperparathyroid changes. In contrast to previous reports, no deterioration of renal function attributable to the treatment occurred, perhaps because a modest dose of 1,25(OH)2D3 was employed combined with meticulous monitoring. Further investigation is required to determine whether alternative therapeutic strategies (smaller doses or intermittent therapy) may avoid the potential for suppressing bone turnover to abnormally low levels in the long term

Densidade da madeira de árvores em savanas do norte da Amazônia brasileira

Densidade da madeira (DM) é uma variável importante para estimativas de estoques de carbono arbóreo em ecossistemas terrestres. Este tema é pobremente investigado em áreas de savana da Amazônia brasileira. O objetivo deste estudo foi investigar a DM das oito principais espécies arbóreas que ocorrem na savana aberta de Roraima, a maior área de savana do norte do bioma Amazônia. Foram verificadas as variações na DM em função da espécie e dos diferentes diâmetros observados ao longo da dimensão vertical de 75 indivíduos amostrados em seis sítios de coleta. Foi utilizado o método direto para obtenção de peças de madeira do fuste e da copa. Os resultados indicaram discrepância significativa interespecífica, sendo Roupala montana Aubl. a espécie de maior DM média (0,674 g cm-3). Foi detectado que existe variação significativa da DM entre as peças do fuste e da copa, independente da espécie e do sítio de coleta. A densidade da madeira de peças da copa com diâmetro entre 5 e 10 cm pode ser utilizada como preditora da DM média do indivíduo arbóreo. Nós concluimos que a DM das oito espécies arbóreas investigadas possui variabilidade interespecífica, com discrepâncias entre a DM do fuste e das partes lenhosas da copa. As distinções aqui detectadas devem ser considerados como uma importante ferramenta para melhorar as estimativas de estoque de carbono em áreas de savanas na Amazônia

Sustained VWF‐ADAMTS‐13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction

Background Prolonged recovery is common after acute SARS-CoV-2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS-13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. Patients and methods Fifty patients were reviewed at a minimum of 6 weeks following acute COVID-19. ADAMTS-13, Weibel Palade Body (WPB) proteins, and angiogenesis-related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID-19 patients (n = 36). Results ADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. Conclusion Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence

ADAMTS13 regulation of VWF multimer distribution in severe COVID‐19

Background Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID-19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS-13)--mediated proteolysis. Objectives This study investigated the hypothesis that ADAMTS-13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection contributing to the observed microvascular thrombosis. Patients and Methods Patients with COVID-19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS-13 activity, and known inhibitors thereof were assessed. Results We observed markedly increased VWF collagen-binding activity in patients with severe COVID-19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS-13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS-13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID-19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS-13 and other proteases. We observed that both N- and O-linked sialylation were altered in severe COVID-19. Furthermore, plasma levels of the ADAMTS-13 inhibitors interleukin-6, thrombospondin-1, and platelet factor 4 were significantly elevated. Conclusions These findings support the hypothesis that SARS-CoV-2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down-regulation in ADAMTS-13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS-13-VWF multimer dysfunction may be useful in COVID-microvascular thrombosis and angiopathy

Common Genetic Variation And Age at Onset Of Anorexia Nervosa

Background Genetics and biology may influence the age at onset of anorexia nervosa (AN). The aims of this study were to determine whether common genetic variation contributes to AN age at onset and to investigate the genetic associations between age at onset of AN and age at menarche. Methods A secondary analysis of the Psychiatric Genomics Consortium genome-wide association study (GWAS) of AN was performed which included 9,335 cases and 31,981 screened controls, all from European ancestries. We conducted GWASs of age at onset, early-onset AN (< 13 years), and typical-onset AN, and genetic correlation, genetic risk score, and Mendelian randomization analyses. Results Two loci were genome-wide significant in the typical-onset AN GWAS. Heritability estimates (SNP-h2) were 0.01-0.04 for age at onset, 0.16-0.25 for early-onset AN, and 0.17-0.25 for typical-onset AN. Early- and typical-onset AN showed distinct genetic correlation patterns with putative risk factors for AN. Specifically, early-onset AN was significantly genetically correlated with younger age at menarche, and typical-onset AN was significantly negatively genetically correlated with anthropometric traits. Genetic risk scores for age at onset and early-onset AN estimated from independent GWASs significantly predicted age at onset. Mendelian randomization analysis suggested a causal link between younger age at menarche and early-onset AN. Conclusions Our results provide evidence consistent with a common variant genetic basis for age at onset and implicate biological pathways regulating menarche and reproduction.Peer reviewe