Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been ...proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.
Cannabis use in pregnancy is associated with adverse perinatal outcomes, which are likely mediated by the placenta. However, the underlying mechanisms and specific vasoactive effects of cannabis on ...the placenta are unknown. Our objective was to determine the impact of chronic prenatal delta-tetrahydrocannabinol (THC, main psychoactive component of cannabis) exposure on placental function and development in a rhesus macaque model using advanced imaging. Animals were divided into two groups, control (CON, n = 5) and THC-exposed (THC, n = 5). THC-exposed animals received a THC edible daily pre-conception and throughout pregnancy. Animals underwent serial ultrasound and MRI at gestational days 85 (G85), G110, G135 and G155 (full term is ~ G168). Animals underwent cesarean delivery and placental collection at G155 for histologic and RNA-Seq analysis. THC-exposed pregnancies had significantly decreased amniotic fluid volume (p < 0.001), placental perfusion (p < 0.05), and fetal oxygen availability (p < 0.05), all indicators of placental insufficiency. Placental histological analysis demonstrated evidence of ischemic injury with microinfarctions present in THC-exposed animals only. Bulk RNA-seq demonstrated that THC alters the placental transcriptome and pathway analysis suggests dysregulated vasculature development and angiogenesis pathways. The longer-term consequences of these adverse placental findings are unknown, but they suggest that use of THC during pregnancy may deleteriously impact offspring development.
Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic ...congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio OR = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10−5). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ∼5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ∼4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias.
Approximately 20% of patients hospitalized for COPD exacerbations in the United States will be readmitted within 30 days. The Centers for Medicare and Medicaid Services has recently proposed to ...revise the Hospital Readmissions Reduction Program to financially penalize hospitals with high all-cause 30-day rehospitalization rates after a hospitalization for COPD exacerbation on or after October 1, 2014.
To report the results of a systematic review of randomized clinical trials evaluating interventions to reduce the rehospitalizations after COPD exacerbations.
Multiple electronic databases were systematically searched to identify relevant studies published between January 1966 and June 2013. Titles, abstracts, and, subsequently, full-text articles were assessed for eligibility. Each study was appraised using predefined criteria.
Among 913 titles and abstracts screened, 5 studies (1,393 participants) met eligibility criteria. All studies had a primary outcome of rehospitalization at 6 or 12 months. No study examined 30-day rehospitalization as the primary outcome. Each study tested a different set of interventions. Two studies (one conducted in Canada and one conducted in Spain and Belgium) showed a decrease in all-cause rehospitalization over 12 months in the intervention group versus comparator group (mean number of hospitalizations per patient, 1.0 vs. 1.8; P = 0.01; percent hospitalized, 45 vs. 67%; P = 0.028; respectively). The only study conducted in the United States found a greater than twofold higher risk of mortality in the intervention group (17 vs. 7%, P = 0.003) but no significant difference in rehospitalizations. It was unclear which set of interventions was effective or harmful.
The evidence base is inadequate to recommend specific interventions to reduce rehospitalizations in this population and does not justify penalizing hospitals for high 30-day rehospitalization rates after COPD exacerbations.
Hypoxia commonly occurs within tumours and is a major cause of radiotherapy resistance. Clinical outcomes could be improved by locating and selectively increasing the dose delivered to hypoxic ...regions. Here we describe a miniature implantable sensor for real-time monitoring of tissue oxygenation that could enable this novel treatment approach to be implemented. The sensor uses a solid-state electrochemical cell that was microfabricated at wafer level on a silicon substrate, and includes an integrated reference electrode and electrolyte membrane. It gave a linear response to oxygen concentration, and was unaffected by sterilisation and irradiation, but showed susceptibility to biofouling. Oxygen selectivity was also evaluated against various clinically relevant electroactive compounds. We investigated its robustness and functionality under realistic clinical conditions using a sheep model of lung cancer. The sensor remained functional following CT-guided tumour implantation, and was sufficiently sensitive to track acute changes in oxygenation within tumour tissue.
•We describe a miniature implantable oxygen sensor to improve radiotherapy treatment•The sensor was microfabricated on silicon using novel wafer-level processes•Electrochemical oxygen detection was performed using the fully integrated sensor•Measurements of lung tumour oxygenation were made in a large animal model
Sepsis is a highly heterogeneous syndrome, which has hindered the development of effective therapies. This has prompted investigators to develop a precision medicine approach aimed at identifying ...biologically homogenous subgroups of patients with septic shock and critical illnesses. Transcriptomic analysis can identify subclasses derived from differences in underlying pathophysiological processes that may provide the basis for new targeted therapies. The goal of this study was to elucidate pathophysiological pathways and identify pediatric septic shock subclasses based on whole blood RNA expression profiles.
The subjects were critically ill children with cardiopulmonary failure who were a part of a prospective randomized insulin titration trial to treat hyperglycemia. Genome-wide expression profiling was conducted using RNA sequencing from whole blood samples obtained from 46 children with septic shock and 52 mechanically ventilated noninfected controls without shock. Patients with septic shock were allocated to subclasses based on hierarchical clustering of gene expression profiles, and we then compared clinical characteristics, plasma inflammatory markers, cell compositions using GEDIT, and immune repertoires using Imrep between the two subclasses.
Patients with septic shock depicted alterations in innate and adaptive immune pathways. Among patients with septic shock, we identified two subtypes based on gene expression patterns. Compared with Subclass 2, Subclass 1 was characterized by upregulation of innate immunity pathways and downregulation of adaptive immunity pathways. Subclass 1 had significantly worse clinical outcomes despite the two classes having similar illness severity on initial clinical presentation. Subclass 1 had elevated levels of plasma inflammatory cytokines and endothelial injury biomarkers and demonstrated decreased percentages of CD4 T cells and B cells and less diverse T cell receptor repertoires.
Two subclasses of pediatric septic shock patients were discovered through genome-wide expression profiling based on whole blood RNA sequencing with major biological and clinical differences. Trial Registration This is a secondary analysis of data generated as part of the observational CAF-PINT ancillary of the HALF-PINT study (NCT01565941). Registered March 29, 2012.
During biosynthesis, Factor VIII (FVIII) undergoes complex post-translational modification including significant glycosylation. Consequently each FVIII molecule can contains 25 N- and 6 O-linked ...glycans. These carbohydrate structures are of physiological significance. For example, FVIII glycan expression modulates intracellular trafficking and also regulates FVIII clearance by dendritic cells. Nevertheless, the molecular mechanisms through which glycan structures influence FVIII biology remains poorly defined. Interestingly, carbohydrate-binding galectins (Gal) -1 and -3 have recently been reported to bind human VWF. Moreover, these galectin interactions significantly influence VWF function. In this study, based upon similar glycans expression profiles, we hypothesised that galectins might also constitute novel binding partners for human FVIII.
In brief, His-tagged Gal-1 and Gal-3 were expressed in E-coli and purified using nickel chromatography. Recombinant FVIII (rFVIII) was purified from different commercial concentrates. Subsequently, FVIII glycosylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. Galectin-FVIII interaction was characterised using modified immunosorbant assays and surface plasmon resonance (SPR).
In plate–binding assays using purified proteins and SPR studies, both Gal-1 and Gal-3 bound to full length rFVIII in a time- and dose-dependent manner. Interestingly the apparent affinities of the galectin-FVIII interactions (Kd of 0.11 ± 0.02nM for Gal-1 and 0.21 ± 0.1nM for Gal-3 respectively) were unusually high for these lectins. Digestion with PNGase F to remove N-linked glycans ablated FVIII binding to Gal-1 (8.6 ± 1%; p<0.0001). In contrast, PNG-FVIII retained significant ability to bind Gal-3 (30.3 ± 3%; p<0.0001). However, combined FVIII digestion with both PNGase F and O glycosidase further attenuated Gal-3 binding (16.5 ± 2%; p<0.05). Cumulatively these findings suggest that whilst Gal-1 binding is mediated predominantly through the N-linked glycans of FVIII, both N- and O-linked glycans modulate its interaction with Gal-3.
The majority of FVIII glycans are contained within the B domain. Unsurprisingly, Gal-1 and Gal-3 binding were both markedly attenuated for B domain deleted rFVIII compared to full length rFVIII (42 ± 1% and 26 ± 0.8%; p<0.0001). Previous studies have described different glycosylation profiles for specific full length commercial rFVIII products. To investigate the relevance of this differential glycosylation, we compared the galectin-binding properties of Advate® (CHO cell line) and Helixate® (BHK cell line). Interestingly, Gal-1 and Gal-3 both displayed significantly enhanced affinity for Helixate (107 ± 2% and 124 ± 1%; p<0.05). These findings are consistent with the fact that the N-linked glycans of BHK-derived FVIII express galactose α1-3 galactose epitopes which constitute preferential galectin-binding ligands.
To determine whether FVIII interacts with galectins in vivo, immunoprecipitation studies were performed using plasma from VWF-/- mice. We observed that that both Gal-1 and Gal-3 were co-precipitated with FVIII even in the absence of VWF. Consequently, both the VWF-FVIII complex and free FVIII in plasma are likely to circulate in a complex with galectins. Importantly, recent studies have reported a prothrombotic phenotype in Gal-1/Gal-3 double deficient mice compared to wild type controls following ferric chloride injury. To investigate whether galectin-binding influences FVIII function, FVIII activity was assessed using a one-stage clotting assay in the presence of increasing galectin concentrations. Interestingly, preincubation of FVIII with Gal-1 (0.5-17µM) resulted in a significant dose-dependent prolongation of the APTT (58 ± 0.2 sec compared to 26 ± 3 secs, p<0.001) In contrast, no such effect was observed for galectin-3 up to 20µM, suggesting these galectins may have differential effects on FVIII biology.
In conclusion, we identify Gal-1 and Gal-3 as novel direct ligands for human FVIII. Both the N- and O-linked carbohydrates of FVIII contribute to galectin binding. Importantly, different commercial FVIII concentrates do not interact with galectins in the same manner. Finally, we also demonstrate that plasma FVIII can circulate in complex with both Gal-1 and Gal-3, and that Gal-1 binding can inhibit the procoagulant function of FVIII.
No relevant conflicts of interest to declare.
The Palomar Transient Factory (PTF) is systematically charting the optical transient and variable sky. A primary science driver of PTF is building a complete inventory of transients in the local ...universe (distance less than 200 Mpc). Here, we report the discovery of PTF 10fqs, a transient in the luminosity 'gap' between novae and supernovae. Located on a spiral arm of Messier 99, PTF 10fqs has a peak luminosity of Mr = --12.3, red color (g -- r = 1.0), and is slowly evolving (decayed by 1 mag in 68 days). It has a spectrum dominated by intermediate-width H Delta *a (930 km s--1) and narrow calcium emission lines. The explosion signature (the light curve and spectra) is overall similar to that of M85 OT2006-1, SN 2008S, and NGC 300 OT. The origin of these events is shrouded in mystery and controversy (and in some cases, in dust). PTF 10fqs shows some evidence of a broad feature (around 8600 A) that may suggest very large velocities (10,000 km s--1) in this explosion. Ongoing surveys can be expected to find a few such events per year. Sensitive spectroscopy, infrared monitoring, and statistics (e.g., disk versus bulge) will eventually make it possible for astronomers to unravel the nature of these mysterious explosions.
There is a nationally recognized shortage of pharmacists in the United States that is more pronounced in the West. This shortage will worsen with the rapid growth and aging of the population. Demand ...for traditional pharmaceutical care services has grown faster in the last decade than predicted and is projected to outpace the entry of pharmacists into the workforce. The growth of direct patient care roles for pharmacists, together with the increasing need for pharmacist services in the management of health care, threatens to overpower the present capacity of pharmaceutical education. Healthcare officials and consumers alike are becoming increasingly concerned about the pressure on pharmacist services leading to decreased access to pharmaceutical care and an increase in the frequency of medication errors both having a major impact on the health of patients. While the impact of the pharmacist shortage on the quality of pharmaceutical care has been the subject of recent concern, the role of pharmacists in economic development has not been adequately addressed. In this review, we examine the impact of the shortage of pharmacists and apply economic principals to the impact created by the addition to a state economy of a public school of pharmacy. We apply this analysis to the proposed addition of a public school of pharmacy in Nevada, the fastest growing state in the nation, which currently has a single, private pharmacy school.
The Palomar Transient Factory (PTF) is systematically charting the optical transient and variable sky. A primary science driver of PTF is building a complete inventory of transients in the local ...Universe (distance less than 200 Mpc). Here, we report the discovery of PTF10fqs, a transient in the luminosity "gap" between novae and supernovae. Located on a spiral arm of Messier 99, PTF 10fqs has a peak luminosity of Mr = -12.3, red color (g-r = 1.0) and is slowly evolving (decayed by 1 mag in 68 days). It has a spectrum dominated by intermediate-width H (930 km/s) and narrow calcium emission lines. The explosion signature (the light curve and spectra) is overall similar to thatof M85OT2006-1, SN2008S, and NGC300OT. The origin of these events is shrouded in mystery and controversy (and in some cases, in dust). PTF10fqs shows some evidence of a broad feature (around 8600A) that may suggest very large velocities (10,000 km/s) in this explosion. Ongoing surveys can be expected to find a few such events per year. Sensitive spectroscopy, infrared monitoring and statistics (e.g. disk versus bulge) will eventually make it possible for astronomers to unravel the nature of these mysterious explosions.
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