ABSTRACT
Mutation detection through exome sequencing allows simultaneous analysis of all coding sequences of genes. However, it cannot yet replace Sanger sequencing (SS) in diagnostics because of ...incomplete representation and coverage of exons leading to missing clinically relevant mutations. Targeted next‐generation sequencing (NGS), in which a selected fraction of genes is sequenced, may circumvent these shortcomings. We aimed to determine whether the sensitivity and specificity of targeted NGS is equal to those of SS. We constructed a targeted enrichment kit that includes 48 genes associated with hereditary cardiomyopathies. In total, 84 individuals with cardiomyopathies were sequenced using 151 bp paired‐end reads on an Illumina MiSeq sequencer. The reproducibility was tested by repeating the entire procedure for five patients. The coverage of ≥30 reads per nucleotide, our major quality criterion, was 99% and in total ∼21,000 variants were identified. Confirmation with SS was performed for 168 variants (155 substitutions, 13 indels). All were confirmed, including a deletion of 18 bp and an insertion of 6 bp. The reproducibility was nearly 100%. We demonstrate that targeted NGS of a disease‐specific subset of genes is equal to the quality of SS and it can therefore be reliably implemented as a stand‐alone diagnostic test.
A NGS stand‐alone diagnostic test for hereditary cardio‐myopathies. Parallel analyses of 48 genes on a MiSeq identified ∼21,000 variants in 84 patients. The sensitivity and specificity of this test equals Sanger Sequencing. In total 168 variants were confirmed, no false‐positives or false‐negatives were detected.
The Soil Moisture Active Passive (SMAP) mission Level-4 Surface and Root-Zone Soil Moisture (L4_SM) data product is generated by assimilating SMAP L-band brightness temperature observations into the ...NASA Catchment land surface model. The L4_SM product is available from 31 March 2015 to present (within 3 days from real time) and provides 3-hourly, global, 9-km resolution estimates of surface (0–5 cm) and root-zone (0–100 cm) soil moisture and land surface conditions. This study presents an overview of the L4_SM algorithm, validation approach, and product assessment versus in situ measurements. Core validation sites provide spatially averaged surface (root zone) soil moisture measurements for 43 (17) “reference pixels” at 9- and 36-km gridcell scales located in 17 (7) distinct watersheds. Sparse networks provide point-scale measurements of surface (root zone) soil moisture at 406 (311) locations. Core validation site results indicate that the L4_SM product meets its soil moisture accuracy requirement, specified as an unbiased RMSE (ubRMSE, or standard deviation of the error) of 0.04 m³ m−3 or better. The ubRMSE for L4_SM surface (root zone) soil moisture is 0.038 m³ m−3 (0.030 m³ m−3) at the 9-km scale and 0.035 m³ m−3 (0.026 m³ m−3) at the 36-km scale. The L4_SM estimates improve (significantly at the 5% level for surface soil moisture) over model-only estimates, which do not benefit from the assimilation of SMAP brightness temperature observations and have a 9-km surface (root zone) ubRMSE of 0.042 m³ m−3 (0.032 m³ m−3). Time series correlations exhibit similar relative performance. The sparse network results corroborate these findings over a greater variety of climate and land cover conditions.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The National Aeronautics and Space Administration Soil Moisture Active Passive (SMAP) mission has been validating its soil moisture (SM) products since the start of data production on March 31, 2015. ...Prior to launch, the mission defined a set of criteria for core validation sites (CVS) that enable the testing of the key mission SM accuracy requirement (unbiased root-mean-square error <0.04 m 3 /m 3 ). The validation approach also includes other ("sparse network") in situ SM measurements, satellite SM products, model-based SM products, and field experiments. Over the past six years, the SMAP SM products have been analyzed with respect to these reference data, and the analysis approaches themselves have been scrutinized in an effort to best understand the products' performance. Validation of the most recent SMAP Level 2 and 3 SM retrieval products (R17000) shows that the L -band (1.4 GHz) radiometer-based SM record continues to meet mission requirements. The products are generally consistent with SM retrievals from the European Space Agency Soil Moisture Ocean Salinity mission, although there are differences in some regions. The high-resolution (3-km) SM retrieval product, generated by combining Copernicus Sentinel-1 data with SMAP observations, performs within expectations. Currently, however, there is limited availability of 3-km CVS data to support extensive validation at this spatial scale. The most recent (version 5) SMAP Level 4 SM data assimilation product providing surface and root-zone SM with complete spatio-temporal coverage at 9-km resolution also meets performance requirements. The SMAP SM validation program will continue throughout the mission life; future plans include expanding it to forested and high-latitude regions.
A ruthenium oxide (RuOx) electrode was used to monitor contractile events of human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) through electrical impedance spectroscopy (EIS). Using RuOx ...electrodes presents an advantage over standard thin film Pt electrodes because the RuOx electrodes can also be used as electrochemical sensor for pH, O
, and nitric oxide, providing multisensory functionality with the same electrode. First, the EIS signal was validated in an optically transparent well-plate setup using Pt wire electrodes. This way, visual data could be recorded simultaneously. Frequency analyses of both EIS and the visual data revealed almost identical frequency components. This suggests both the EIS and visual data captured the similar events of the beating of (an area of) hPSC-CMs. Similar EIS measurement was then performed using the RuOx electrode, which yielded comparable signal and periodicity. This mode of operation adds to the versatility of the RuOx electrode's use in in vitro studies.
Abstract SET domain-containing 2 ( SETD2 ) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma ...(ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1 , whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.
studies which focus on cellular metabolism can benefit from time-resolved readouts from the living cells. pH and O
concentration are fundamental parameters upon which cellular metabolism is often ...inferred. This work demonstrates a novel use of a ruthenium oxide (RuO
) electrode for
studies. The RuO
electrode was characterized to measure both pH and O
using two different modes. When operated potentiometrically, continuous pH reading can be obtained, and O
concentration can be measured chronoamperometrically. In this work, we demonstrate the use of the RuO
electrodes in inferring two different types of metabolism of human pluripotent stem cell-derived cardiomyocytes. We also show and discuss the interpretation of the measurements into meaningful extracellular acidification rates and oxygen consumption rates of the cells. Overall, we present the RuO
electrode as a versatile and powerful tool in
cell metabolism studies, especially in comparative settings.
Three independent surface soil moisture datasets for the period 1979–87 are compared: 1) global retrievals from the Scanning Multichannel Microwave Radiometer (SMMR), 2) global soil moisture derived ...from observed meteorological forcing using the NASA Catchment Land Surface Model, and 3) ground-based measurements in Eurasia and North America from the Global Soil Moisture Data Bank. Time-average soil moisture fields from the satellite and the model largely agree in the global patterns of wet and dry regions. Moreover, the time series and anomaly time series of monthly mean satellite and model soil moisture are well correlated in the transition regions between wet and dry climates where land initialization may be important for seasonal climate prediction. However, the magnitudes of time-average soil moisture and soil moisture variability are markedly different between the datasets in many locations. Absolute soil moisture values from the satellite and the model are very different, and neither agrees better with ground data, implying that a “correct” soil moisture climatology cannot be identified with confidence from the available global data. The discrepancies between the datasets point to a need for bias estimation and correction or rescaling before satellite soil moisture can be assimilated into land surface models.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their ...associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.
A new method for quantitative etching of the poly(dimethylsiloxane) block in polystyrene−poly(dimethylsiloxane) (PS−PDMS) block copolymers is reported. Reacting the block copolymer with anhydrous ...hydrogen fluoride renders a nanoporous material (NPM) with the remaining glassy PS maintaining the original bulk morphology. 1H NMR, mass difference, size exclusion chromatography, and X-ray photoelectron spectroscopy were used to characterize the materials before and after etching. NPMs containing spherical and gyroid cavities were prepared, as ascertained by small-angle X-ray scattering. This is the first report on block copolymer-based NPM films of millimeter thickness containing secluded spherical holes. Surface images by AFM and SEM are consistent with the SAXS findings.
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