Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

Background: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR) and multiple annealing and looping-based amplification cycles (MALBAC). However, a comprehensive comparison of variations detection performance between these WGA methods has not yet been performed. Results: We systematically compared the advantages and disadvantages of different WGA methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly higher genome recovery sensitivity (~84 %) than DOP-PCR (~6 %) and MALBAC (~52 %) at high sequencing depth. MALBAC and MDA had comparable single-nucleotide variations detection efficiency, false-positive ratio, and allele drop-out ratio. We further demonstrated that SCRS data amplified by either MDA or MALBAC from a gastric cancer cell line could accurately detect gastric cancer CNVs with comparable sensitivity and specificity, including amplifications of 12p11.22 (KRAS) and 9p24.1 (JAK2, CD274, and PDCD1LG2). Conclusions: Our findings provide a comprehensive comparison of variations detection performance using SCRS amplified by different WGA methods. It will guide researchers to determine which WGA method is best suited to individual experimental needs at single-cell level

Frequent alterations in cytoskeleton remodelling genes in primary and metastatic lung adenocarcinomas

The landscape of genetic alterations in lung adenocarcinoma derived from Asian patients is largely uncharacterized. Here we present an integrated genomic and transcriptomic analysis of 335 primary lung adenocarcinomas and 35 corresponding lymph node metastases from Chinese patients. Altogether 13 significantly mutated genes are identified, including the most commonly mutated gene TP53 and novel mutation targets such as RHPN2, GLI3 and MRC2. TP53 mutations are furthermore significantly enriched in tumours from patients harbouring metastases. Genes regulating cytoskeleton remodelling processes are also frequently altered, especially in metastatic samples, of which the high expression level of IQGAP3 is identified as a marker for poor prognosis. Our study represents the first large-scale sequencing effort on lung adenocarcinoma in Asian patients and provides a comprehensive mutational landscape for both primary and metastatic tumours. This may thus form a basis for personalized medical care and shed light on the molecular pathogenesis of metastatic lung adenocarcinoma

Peripheral Deformation and Buckling of Stainless Steel Hemispherical Shells Compressed by a Flat Plate

Abstract An experimental investigation was performed on stainless steel hemispherical shells under axial compression. Eight kinds of shells with radius-to-thickness ratios that range from 57.1 to 125 were designed and manufactured for this study. The shells were compressed to more than 50% of their radii by a solid flat plate. To avoid contact between the base plate and the deformed central part of the shells, most of the shells were placed on a plate with a hole in the center. Nonetheless, one type of shell was placed on a solid base plate without a hole to analyze the effect of the base plate. As per an observation of collapse modes and load-deformation shell relations, the deformation process of a hemispherical shell that is compressed by a flat plate can be divided into four stages: local flattening (Stage I), axi-symmetric inward dimpling (Stage II), non-symmetric multiple lobes (Stage III), and peripheral deformation and buckling stage (Stage IV). The present study mainly studies Stage IV, which can be categorized into peripheral compression (Stage A), peripheral buckling (Stage B), buckling expanding (Stage C), and overall collapse (Stage D)

Investigation on Roles of Packing Density and Water Film Thickness in Synergistic Effects of Slag and Silica Fume

The ternary blended cement with finer slag and silica fume (SF) could improve the packing density (PD) through the filling effect. The excess water (water more than needed for filling into voids between the cement particles) can be released to improve the fresh properties and densify the microstructure which is beneficial for improving the hardened properties. To verify the hypothesis and reveal how and why (cement + slag + SF) the ternary blends could bring such advantages, the binder pastes incorporating slag and SF with various water-to-binder ratios were produced to determine the PD experimentally. To evaluate the optimum water demand (OWD) for maximum wet density, the influence of the dispersion state of the binder on PD was investigated using the wet packing density approach. The effect of PD of various binary and ternary binder systems on water film thickness (WFT), fluidity, setting time, and compressive strength development of cement paste was also investigated. The results show that the ternary blends could improve the PD and decrease the water film thickness (WFT). The enhanced PD and altered WFT are able to increase fluidity and compressive strength. The ternary blends could improve the compressive strengths by increasing PD and exerting nucleation and pozzolanic effects

Serum Exosomal MicroRNAs as Potential Circulating Biomarkers for Endometriosis

Background. A reliable noninvasive biomarker is not yet available for endometriosis diagnosis. Novel biomarkers for the diagnosis of endometriosis are urgently needed. The molecular constituents of exosomes, especially exosomal microRNAs (miRNAs), have considerable potential as novel biomarkers for clinical diagnosis. This study is aimed at exploring aberrant exosomal miRNA profiles by using miRNA microarray and at providing more accurate molecular biomarkers of endometriosis. Methods. Exosomes were isolated from the serum of patients with endometriosis and negative controls and identified by electron microscopy, nanoparticle tracking analysis, and Western blot. Exosomal miRNAs were profiled by miRNA microarrays. The expression of selective serum exosomal miRNA was validated by qRT-PCR. Receiver operating characteristic (ROC) curves were established to explore the diagnostic value of selective miRNAs. Finally, GO annotation and KEGG pathway enrichment analyses were used to display possible functions associated with the two miRNAs. Results. A total of 24 miRNAs showed differential levels of enrichment with P1 by miRNA microarrays. Among the six selective miRNAs (i.e., miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, and miR-939-5p), qRT-PCR analysis revealed that miR-22-3p and miR-320a were significantly upregulated in serum exosomes from patients with endometriosis compared with negative individuals. ROC curve revealed that the serum exosomal miR-22-3p and miR-320a yielded the area under the curve values of 0.855 and 0.827, respectively. Conclusion. Our results demonstrated that exosomal miR-22-3p and miR-320a were significantly increased in the sera of patients with endometriosis. The two miRNAs may be useful potential biomarkers for endometriosis diagnosis

Research on the Mechanical Behavior of Buried Double-Wall Corrugated Pipes

The mechanical behavior of buried HDPE double-wall corrugated pipes is mainly affected by the material and the structure of the pipe wall. Here we studied a peculiar material that added fly ash (FA) in high density polyethylene (HDPE) to develop composites. We have conducted research on FA/HDPE composites with different mix proportions. When 5% compatibilizer was added to the 10% FA masterbatch/HDPE composite, the Young’s Modulus of FA/HDPE composite was higher. This paper mainly studies the mechanical behavior of the structure of pipe walls for materials with this proportion of the ingredients. The mechanical behavior of double-wall corrugated pipes with different ratios of interior and exterior wall thicknesses is studied by keeping the sum of the interior and exterior wall thicknesses unchanged. Pipes with six different ratios of interior and exterior wall thicknesses are simulated; the results show that the strain of crest and liner gradually decreased and the valley strain gradually increased with the increase of the exterior wall thickness. By comparing inner and outer wall thickness ratios from 0.67 to 2.33, it is found that the structural performance and economic advantage for the double-wall corrugated pipes is best when the thickness ratio of the interior wall and the exterior wall is controlled to be from 1.3 to 1.8. This paper expounds the deformation mechanism of double-wall corrugated pipes from the perspective of mechanical behavior and structural characteristics, and provides a reference for material selection and structural design of double-wall corrugated pipes

Preparation and Finite Element Analysis of Fly Ash/HDPE Composites for Large Diameter Bellows

In recent years, buried bellows have often had safety accidents such as pipeline bursts and ground subsidence due to the lack of adequate mechanical properties and other quality problems. In order to improve the mechanical properties of bellows, fly ash (FA) was used as a reinforced filler in high density polyethylene (HDPE) to develop composites. The FA was surface treated with a silane coupling agent and HDPE-g-maleic anhydride was used as compatibilizer. Dumbbell-shaped samples were prepared via extrusion blending and injection molding. The cross-section morphology, thermal stability and mechanical properties of the composites were studied. It was observed that when 10% modified FA and 5% compatibilizer were added to HDPE, the tensile yield strength and tensile breaking strength of the composites were nearly 30.2% and 40.4% higher than those of pure HDPE, respectively, and the Young’s modulus could reach 1451.07 MPa. In addition, the ring stiffness of the bellows was analyzed using finite element analysis. Compared with a same-diameter bellows fabricated from common commercially available materials, the ring stiffness increased by nearly 23%. The preparation method of FA/HDPE is simple, efficient, and low-cost. It is of great significance for the popularization of high-performance bellows and the high value-added utilization of FA

Multiplexed Single-Cell Leukocyte Enzymatic Secretion Profiling from Whole Blood Reveals Patient-Specific Immune Signature

© 2021 American Chemical Society. Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 μL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers