The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative ...capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene-therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, peptide-modified derivative of wild-type AAV9 (AAV9_A2) and peptide-modified derivative of wild-type AAV1 (AAV1_P5), both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of the promoter and dose of injected viral genomes enabled labeling of 30%–60% of the NSC compartment, which was validated by fluorescence-activated cell sorting (FACS) analyses and single-cell RNA sequencing. Importantly, transduced NSCs readily produced neurons. The present study identifies AAV variants with a high regional tropism toward the ventricular-subventricular zone (v-SVZ) with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.
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High-throughput screening of 177 AAV capsid variants identifies variants with high tropism for the adult subventricular zone. Single-cell transcriptomics shows that lineages arising from transduced stem cells are comparable to non-transduced ones. Mathematical modeling and flow cytometry indicate transduction of 30%−60% stem cells within the subventricular zone.
We present an overview of the National Science Foundation’s
Daniel K. Inouye Solar Telescope
(DKIST), its instruments, and support facilities. The 4 m aperture DKIST provides the highest-resolution ...observations of the Sun ever achieved. The large aperture of DKIST combined with state-of-the-art instrumentation provide the sensitivity to measure the vector magnetic field in the chromosphere and in the faint corona, i.e. for the first time with DKIST we will be able to measure and study the most important free-energy source in the outer solar atmosphere – the coronal magnetic field. Over its operational lifetime DKIST will advance our knowledge of fundamental astronomical processes, including highly dynamic solar eruptions that are at the source of space-weather events that impact our technological society. Design and construction of DKIST took over two decades. DKIST implements a fast (f/2), off-axis Gregorian optical design. The maximum available field-of-view is 5 arcmin. A complex thermal-control system was implemented in order to remove at prime focus the majority of the 13 kW collected by the primary mirror and to keep optical surfaces and structures at ambient temperature, thus avoiding self-induced local seeing. A high-order adaptive-optics system with 1600 actuators corrects atmospheric seeing enabling diffraction limited imaging and spectroscopy. Five instruments, four of which are polarimeters, provide powerful diagnostic capability over a broad wavelength range covering the visible, near-infrared, and mid-infrared spectrum. New polarization-calibration strategies were developed to achieve the stringent polarization accuracy requirement of 5×10
−4
. Instruments can be combined and operated simultaneously in order to obtain a maximum of observational information. Observing time on DKIST is allocated through an open, merit-based proposal process. DKIST will be operated primarily in “service mode” and is expected to on average produce 3 PB of raw data per year. A newly developed data center located at the NSO Headquarters in Boulder will initially serve fully calibrated data to the international users community. Higher-level data products, such as physical parameters obtained from inversions of spectro-polarimetric data will be added as resources allow.
Left atrial (LA) low-voltage areas (LVAs) are frequently observed in patients with atrial fibrillation (AF) and may predict AF recurrence after catheter ablation.
The aim of this study was to develop ...and validate a clinical tool to identify LVAs that are associated with AF recurrence after pulmonary vein isolation (PVI).
In a cohort of 238 patients, voltage maps were created during LA procedures. LVAs were defined as areas with electrogram amplitudes <0.5 mV. On the basis of regression analysis, predictors of LA substrate were identified. These parameters were used to establish a dedicated risk score (DR-FLASH score, based on diabetes mellitus, renal dysfunction, persistent form of AF, LA diameter >45 mm, age >65 years, female sex, and hypertension). This risk score was then prospectively validated in a multicenter cohort of 180 patients. The association of the score with long-term recurrence of atrial arrhythmias after circumferential PVI was tested in a retrospective cohort of 484 patients.
The DR-FLASH score effectively identified LVA substrate (C statistic = 0.801, P < .001). In the prospective multicenter validation cohort, the predictive value of the DR-FLASH score was confirmed (C statistic = 0.767, P < .001). The probability for the presence of LA substrate increased by a factor of 2.2 (95% confidence interval CI 1.6-2.9, P < .001) with each point scored. Furthermore, the risk of AF recurrence after PVI increased by a factor of 1.3 (95% CI 1.1-1.5, P < .001) with every additional point and was almost 2 times higher in patients with a DR-FLASH score >3 (odds ratio 1.7, 95% CI 1.1-2.8, P = .026).
The DR-FLASH score may be useful to identify patients who may require extensive substrate modification instead of PVI alone.
The aim of the study was to compare azathioprine versus mesalazine tablets for the prevention of clinical recurrence in patients with postoperative Crohn's disease (CD) with moderate or severe ...endoscopic recurrence.
This was a 1 year, double-blind, double-dummy, randomised study which took place in 21 gastroenterology centres in Austria, the Czech Republic, Germany and Israel. The study participants were 78 adults with CD who had undergone resection with ileocolonic anastomosis in the preceding 6-24 months without subsequent clinical recurrence and with a Crohn's disease activity index (CDAI) score <200, but with moderate or severe endoscopic recurrence. The study drugs were azathioprine 2.0-2.5 mg/kg/day or mesalazine 4 g/day over 1 year. The primary end point was therapeutic failure during 1 year, defined as a CDAI score > or = 200 and an increase of > or = 60 points from baseline, or study drug discontinuation due to lack of efficacy or intolerable adverse drug reaction.
Treatment failure occurred in 22.0% (9/41) of azathioprine-treated patients and 10.8% (4/37) of mesalazine-treated patients, a difference of 11.1% (95% CI -5.0% to 27.3%, p=0.19). Clinical recurrence was significantly less frequent with azathioprine versus mesalazine (0/41 (0%) vs 4/37 (10.8%), p=0.031), whereas study drug discontinuation due to adverse drug reactions only occurred in azathioprine-treated patients (9/41 (22.0%) vs 0%, p=0.002). The proportion of patients showing > or = 1 point reduction in Rutgeerts score between baseline and month 12 was 63.3% (19/30) and 34.4% (11/32) in the azathioprine and mesalazine groups, respectively (p=0.023).
In this population of patients with postoperative CD at high risk of clinical recurrence, superiority for azathioprine versus mesalazine could not be demonstrated for therapeutic failure.
Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the ...methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all data sets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several data sets including E. coli proteome spike-in data, using both label-free and TMT-labeled quantification. Compared with previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared with other statistical methods in both label-free and labeled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.
The value of azathioprine metabolites (6-thioguanine nucleotides 6-TGN) in monitoring clinical treatment response is still controversially discussed. Data regarding thiopurine metabolite levels and ...endoscopic improvement are lacking.
Data were analyzed post hoc from a 1-year, multicenter, double-blind, double-dummy, randomized trial comparing azathioprine 2.0 to 2.5 mg/kg per day versus mesalamine 4 g/d in a subset of 23 postoperative patients with Crohn's disease (CD) treated with azathioprine and having moderate-to-severe endoscopic recurrence according to a modified 6-grade score. Red blood cell (RBC) concentrations of 6-TGN, 6-methyl-mercaptopurine ribonucleotides (6-MMPR), and 6-methyl-thioguanine nucleotides (6-MTGN) were indicated as follows: area under the concentration-time curve, average concentration (C av), and concentration at the final study visit.
Overall, 74% of patients showed an improvement in the modified endoscopic score (P = 0.022). Median endoscopic score reduced from 4 at the baseline to 2 at the final visit. Patients with a high C av for 6-TGN (≥ 193 pmol/8 × 10(8) RBC; P = 0.017) or 6-MTGN (≥ 79.2 pmol/8 × 10(8) RBC; P = 0.035) significantly improved in endoscopic score, and the improvement in endoscopic score correlated with C av for 6-TGN (r = -0.51; P = 0.013). For concentration at the final visit, higher values for 6-TGN (≥ 142 pmol/8 × 10(8) RBC; P = 0.017) were associated with a better postoperative score. Sensitivity analysis revealed a significant correlation between 6-TGN (area under the concentration-time curve) and postoperative endoscopic improvement.
Our post hoc analysis from a double-blind, randomized trial suggests that higher RBC 6-TGN levels are associated with endoscopic improvement in patients with severe postoperative endoscopic recurrence of CD. Thus, our study provides first evidence on the utility of monitoring of thiopurine metabolites to achieve mucosal response in CD.
Compared to whites, blacks have a 2-fold higher incidence of coronary heart disease (CHD), and black race is an independent predictor of worse survival after CHD events after accounting for other ...confounding variables (e.g., socioeconomic, demographic, etc.). However, there has been a paucity of literature considering racial differences in platelet function. We recently reported results from the Platelet RNA And eXpression-1 (PRAX1) study of platelets in healthy black (n=70) and white (n=84) subjects (Edelstein et al, Nat. Med 2013). Human platelets express two thrombin receptors, protease activated receptor (PAR) 1 and PAR4. We demonstrated 1) a 3.7-fold increased PAR4-mediated aggregation kinetics and greater calcium mobilization in platelets from black subjects compared to whites, and 2) phosphatidylcholine transfer protein (PC-TP) was a mediator of this racial difference. These findings have potential clinical significance because greater platelet-mediated thrombosis could contribute to the worse outcomes in blacks than whites after coronary events. Additionally, in the presence of vorapaxar (an FDA-approved PAR1 inhibitor for patients with coronary and peripheral vascular disease) PAR4 is the primary means by which thrombin activates platelets, and the risks and benefits of vorapaxar and other PAR inhibitors by race are unknown.
Because PC-TP expression only accounted for 18% of the observed variance in PAR4 function, we have considered additional mechanisms to explain the racial difference in thrombin-induced PAR4-mediated platelet reactivity. Although platelets from blacks express 14% more PAR4 protein than whites, this difference does not explain the variance in platelet PAR4 function. Genome-wide quantitative trait locus analysis identified common 3 SNPs in the PAR4 gene (F2RL3) as associated with PAR4-induced platelet aggregation (P = 4.79 x 10-11). Importantly, the allele frequency of these SNPs differs by race in both the 1000 Genomes dataset and in PRAX1 (P= 4.31 x 10-16). One of these SNPs, rs773902, determines if residue 120 in PAR4 is an alanine (81% in whites vs. 37% in blacks) or a threonine (63% in blacks vs. 19% in whites). A second less frequent, non-synonymous F2RL3SNP, Phe296Val, was only observed in blacks and was associated with loss of PAR4 function. This variant appears to have a dominant negative effect since only one copy of PAR4-296Val abolished the enhanced PAR4-AP induced platelet aggregation associated with PAR4-Thr120.
A proximal step in PAR4-induced platelet signal transduction is Gq activation leading to hydrolysis of phosphatidylinositol 4,5-biphosphate to IP3 and diacylglycerol, followed by increased cytoplasmic calcium. Platelets with the PAR4-Thr120 variant exhibited a 65% greater Ca2+ flux than PAR4-Ala120 homozygotes (P = 0.03). To eliminate other sources of inter-individual variation we cloned and expressed each variant in 293 cells, which lack endogenous PAR4 expression, and measured the generation of IP3 in response to PAR4-AP treatment. Compared to PAR4-Ala120 expressing cells, PAR4-Thr120 expressing cells generated 41% higher IP3 levels (P = 0.03).
Because PAR inhibitors are either currently approved (vorapaxar) or in development, we examined platelet function in the presence of PAR1 and PAR4 antagonists. PAR4 genotype had no effect on vorapaxar inhibition of PAR1 signaling or PAR4 signaling in the presence of vorapaxar. YD-3, a selective inhibitor of PAR4 and not PAR1, was also tested. In stark contrast to vorapaxar, PAR4 genotype had a significant effect of the efficacy of PAR4 inhibition by the compound YD-3. Platelets from subjects homozygous for the PAR4-Thr120 were not inhibited by any tested concentration of YD-3 while PAR4-Ala120 homozygotes were inhibited in a dose-dependent manner (P = 6x10-8).
In summary, we have identified variation in the PAR4 gene that alters PAR4 function and accounts for 48% of the observed racial difference in platelet PAR4 signaling. These results, couple with other racial data (Tourdot et al., submitted), have two critical clinical implications for anti-platelet therapy: (1) Patients expressing the PAR4-Thr120 variant (mostly black) may not be adequately protected on current anti-platelet drugs, and (2) PAR4 antagonists currently in development may not be effective in PAR4-Thr120 expressing platelets. This suggests a critical need for new and effective therapies for these patients.
No relevant conflicts of interest to declare.
Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an ...especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10-23; q = 10-20) and with PAR4 reactivity (p = 3.4x10-8; q = 3.5x10-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca+ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca+ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10-4; q = 1.38 x10-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34+ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3'UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy.
No relevant conflicts of interest to declare.
The well-known inter-individual variation in human platelet reactivity is heritable, but there is limited understanding of the responsible genetic mechanisms. Prior work supports an effect of age, ...gender, race, body mass index (BMI) and other demographic variables on platelet function, but there is little or no data addressing whether these variables are associated with platelet gene expression. Quantitative and qualitative variants of different classes of platelet RNAs are important windows into megakaryocyte and platelet gene expression, and emerging evidence indicates the utility of specific transcripts as disease biomarkers, as vectors for systemically transmitting genetic information in microparticles, as mediators of pharmacogenetic effects, and as potential therapies. However, to date the small sample sizes of platelet transcriptomic studies have not permitted testing for associations between demographic variables and platelet RNA levels. The Platelet RNA And eXpression-1 (PRAX1) study was designed to characterize mRNAs and microRNAs (miRNAs) in highly purified platelets from a group of healthy, non-diabetic subjects. 163 participants (84 whites and 70 blacks) were recruited, and after exclusion due to use of anti-platelet medication (defined as non-responsiveness to arachidonic acid-induced platelet aggregation) or abnormal hematological parameters, 154 subjects were included for RNA profiling and analyses. Leukocyte-depleted platelet RNA was profiled using the Human Gene 1.0 ST Array (Affymetrix) for mRNA and the nCounter (Nanostring) for miRNA. After normalization, mRNAs and miRNAs were defined as commonly expressed if they were above background in over 65% of subjects. This yielded 5813 common mRNAs and 181 common miRNAs. These profiles were validated using a separate cohort with similar demographic characteristics (n=19) and by plotting rank correlations of mRNAs (r = 0.57; p = 2.3x10-311) and miRNAs (r = 0.69; p = 7.3x10-21). We identified 130 mRNAs and 15 miRNAs that were differentially expressed (DE) by age (Q<0.05). These 130 mRNAs were enriched for putative binding sites for these 15 miRNAs (p<0.001). We identified a network of DE miRNAs targeting DE mRNAs, in which the miRNA and mRNA were significantly and inversely correlated by age. Mitochondrial mRNAs were also inversely correlated with age. Second, we identified 54 mRNAs and 9 miRNAs DE by gender (Q<0.05). As expected, the Y-chromosome genes, EIF1AY, TMSB4Y, UTY and DDX3Y were expressed more highly in males (p = 1.22x10-82, p = 9.28x10-70, p = 2.89x10-68 and p = 7.45x10-58, respectively). A network of miRNAs and mRNAs, both DE by gender, was identified in which the miRNAs were predicted to target the mRNAs. Lastly, a single miRNA but no mRNAs were DE by BMI. In summary, levels of platelet mRNAs and miRNAs are strongly associated with age and gender, but for the most part, not with BMI. The inverse relationship between these two DE RNA classes suggests miRNAs may regulate mRNA levels between genders and upon aging. Future association studies between platelet RNAs and either ex vivo platelet function or in vivo platelet-mediated hemostasis and thrombosis must account for age and gender.
No relevant conflicts of interest to declare.
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