Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA⁺CD41⁺ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA⁺ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.     

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.     

Cytotoxic Potential of a-Azepano- and 3-Amino-3,4-SeCo-Triterpenoids

Semi-synthetic triterpenoids, holding an amino substituted seven-membered A-ring (azepano-ring), which could be synthesized from triterpenic oximes through a Beckmann type rearrangement followed by a reduction of lactame fragment, are considered to be novel promising agents exhibiting anti-microbial, alpha-glucosidase, and butyrylcholinesterase inhibitory activities. In this study, in an attempt to develop new antitumor candidates, a series of A-ring azepano- and 3-amino-3,4-seco-derivatives of betulin, oleanolic, ursolic, and glycyrrhetinic acids were evaluated for their cytotoxic activity against five human cancer cell lines and non-malignant mouse fibroblasts by means of a colorimetric sulforhodamine assay. Azepanoallobetulinic acid amide derivative 11 was the most cytotoxic compound of this series but showed little selectivity between the different human tumor cell lines. Flow cytometry experiments showed compound 11 to act mainly by apoptosis (44.3%) and late apoptosis (21.4%). The compounds were further screened at the National Cancer Institute towards a panel of 60 cancer cell lines. It was found that compounds 3, 4, 7, 8, 9, 11, 15, 16, 19, and 20 showed growth inhibitory (GI₅₀) against the most sensitive cell lines at submicromolar concentrations (0.20–0.94 μM), and their cytotoxic activity (LC₅₀) was also high (1–6 μM). Derivatives 3, 8, 11, 15, and 16 demonstrated a certain selectivity profile at GI₅₀ level from 5.16 to 9.56 towards K-562, CCRF-CEM, HL-60(TB), and RPMI-8226 (Leukemia), HT29 (Colon cancer), and OVCAR-4 (Ovarian cancer) cell lines. Selectivity indexes of azepanoerythrodiol 3 at TGI level ranged from 5.93 (CNS cancer cell lines SF-539, SNB-19 and SNB-75) to 14.89 for HCT-116 (colon cancer) with SI 9.56 at GI₅₀ level for the leukemia cell line K-562. The present study highlighted the importance of A-azepano-ring in the triterpenic core for the development of novel antitumor agents, and a future aim to increase the selectivity profile will thus lie in the area of modifications of azepano-triterpenic acids at their carboxyl group.     

Heteroalleles in Common Wheat: Multiple Differences between Allelic Variants of the Gli-B1 Locus

The Gli-B1-encoded γ-gliadins and non-coding γ-gliadin DNA sequences for 15 different alleles of common wheat have been compared using seven tests: electrophoretic mobility (EM) and molecular weight (MW) of the encoded major γ-gliadin, restriction fragment length polymorphism patterns (RFLPs) (three different markers), Gli-B1-γ-gliadin-pseudogene known SNP markers (Single nucleotide polymorphisms) and sequencing the pseudogene GAG56B. It was discovered that encoded γ-gliadins, with contrasting EM, had similar MWs. However, seven allelic variants (designated from I to VII) differed among them in the other six tests: I (alleles Gli-B1i, k, m, o), II (Gli-B1n, q, s), III (Gli-B1b), IV (Gli-B1e, f, g), V (Gli-B1h), VI (Gli-B1d) and VII (Gli-B1a). Allele Gli-B1c (variant VIII) was identical to the alleles from group IV in four of the tests. Some tests might show a fine difference between alleles belonging to the same variant. Our results attest in favor of the independent origin of at least seven variants at the Gli-B1 locus that might originate from deeply diverged genotypes of the donor(s) of the B genome in hexaploid wheat and therefore might be called “heteroallelic”. The donor’s particularities at the Gli-B1 locus might be conserved since that time and decisively contribute to the current high genetic diversity of common wheat.     

Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 μM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.     

Spectral radiance reconstruction from trichromatic camera responses based on orthogonal test and regularized algorithm

Reconstruction of spectral information based on multi‐channel image system is a significant problem in color reproduction, detection, and recognition. A spectral radiance reconstruction from trichromatic digital camera responses is researched in this article. The mapping relationship between the trichromatic imaging system response and the incident spectral radiance is analyzed. Then, in order to remove the ill‐posedness of the problem, a regularized constraint solution model of spectral radiance reconstruction matrix is established. And the spectral radiance can be reconstructed by spectral radiance reconstruction matrices and trichromatic imaging system response. Finally, the spectral radiance reconstruction matrix is estimated by the system radiometric calibration experiment. The input radiance is offered by a LCD display. A 3‐factor and 9‐level orthogonal test is designed for the calibration experiment, and a test set of 24 colors is used for precision analysis. The results show that the average relative mean error of our method is 8.69%, it is lower than that of Wiener filtering method by 2.84%. The method can reconstruct spectral radiance information effectively.     

Agronomic performance of high oleic,low linolenic soybean in Tennessee

Soybean oil hydrogenation alters the linolenic acid molecule to prevent the oil from becoming rancid, however, health reports have indicated trans-fat caused by hydrogenation, is not generally regarded as safe. Typical soybeans contain approximately 80 g kg⁻¹ to 120 g kg⁻¹ linolenic acid and 240 g kg⁻¹ of oleic acid. In an effort to accommodate the need for high-quality oil, the United Soybean Board introduced an industry standard for a high oleic acid greater than 750 g kg⁻¹ and linolenic acid less than 30 g kg⁻¹ oil. By combing mutations in the soybean plant at four loci, FAD2-1A and FAD2-1B, oleate desaturase genes and FAD3A and FAD3C, linoleate desaturase genes, and seed oil will not require hydrogenation to prevent oxidation and produce high-quality oil. In 2017 and 2018, a study comparing four near-isogenic lines across multiple Tennessee locations was performed to identify agronomic traits associated with mutations in FAD3A and FAD3C loci, while holding FAD2-1A and FAD2-1B constant in the mutant (high oleic) state. Soybean lines were assessed for yield and oil quality based on mutations at FAD2-1 and FAD3 loci. Variations of wild-type and mutant genotypes were compared at FAD3A and FAD3C loci. Analysis using a generalized linear mixed model in SAS 9.4, indicated no yield drag or other negative agronomic traits associated with the high oleic and low linolenic acid genotype. All four mutations of fad2-1A, fad2-1B, fad3A, and fad3C were determined as necessary to produce a soybean with the new industry standard (>750 g kg⁻¹ oleic and <30 g kg⁻¹ linolenic acid) in a maturity group-IV-Late cultivar for Tennessee growers.     

Single-Cell Microgels for Diagnostics and Therapeutics

Cell encapsulation within hydrogel droplets is transforming what is feasible in multiple fields of biomedical science such as tissue engineering and regenerative medicine, in vitro modeling, and cell-based therapies. Recent advances have allowed researchers to miniaturize material encapsulation complexes down to single-cell scales, where each complex, termed a single-cell microgel, contains only one cell surrounded by a hydrogel matrix while remaining <100 μm in size. With this achievement, studies requiring single-cell resolution are now possible, similar to those done using liquid droplet encapsulation. Of particular note, applications involving long-term in vitro cultures, modular bioinks, high-throughput screenings, and formation of 3D cellular microenvironments can be tuned independently to suit the needs of individual cells and experimental goals. In this progress report, an overview of established materials and techniques used to fabricate single-cell microgels, as well as insight into potential alternatives is provided. This focused review is concluded by discussing applications that have already benefited from single-cell microgel technologies, as well as prospective applications on the cusp of achieving important new capabilities.     

虚实结合的可穿戴产品适合性检验研究

目的 为了更科学地研究和检验可穿戴产品的适合性,提出一种适合性检验方法,能够精确保留现实环境中的产品佩戴关系,并能将现实与虚拟的适合性检验研究相结合,得到合理的适合性检验结果.方法 以虚拟现实眼镜的适合性检验为例,通过高精度的三维测量技术将现实环境中的人、产品以及人—产品佩戴关系转化为三维虚拟信息,并以人—产品佩戴三维模型为参考基准对齐人和产品的虚拟模型,得到保留现实佩戴关系的人—产品佩戴模型组,再应用偏差分析法得出人—机佩戴区域的可视化适合性结果和统计数据,结合主观评价方法进一步分析产品的适合性.结论 虚实结合的产品适合性检验方法可在虚拟环境中高精度地保留现实环境中的人—产品佩戴关系,并能得到可视化的适合性检验结果,为检验和指导产品的适合性提供依据.     

keff uncertainty quantification and analysis due to nuclear data during the full lifetime burnup calculation for a small-sized prismatic high temperature gas-cooled reactor

To benefit from recent advances in modeling and computational algorithms,as well as the availability of new covariance data,sensitivity and uncertainty analyses are needed to quantify the impact of uncertain sources on the design parameters of small prismatic high-temperature gas-cooled reactors(HTGRs).In particular,the contribution of nuclear data to the keff uncertainty is an important part of the uncertainty analysis of small-sized HTGR physical calculations.In this study,a small-sized HTGR designed by China Nuclear Power Engineering Co.,Ltd.was selected for keff uncertainty analysis during full lifetime burnup calculations.Models of the cold zero power(CZP)condition and full lifetime burnup process were constructed using the Reactor Monte Carlo Code RMC for neutron transport calculation,depletion calculation,and sensitivity and uncertainty analysis.For the sensitivity analysis,the Contribution-Linked eigenvalue sensitivity/Uncertainty estimation via Track length importance Characterization(CLUTCH)method was applied to obtain sensitive infor-mation,and the"sandwich"method was used to quantify the keff uncertainty.We also compared the keff uncertainties to other typical reactors.Our results show that 235U is the largest contributor to keff uncertainty for both the CZP and depletion conditions,while the contribution of 239Pu is not very significant because of the design of low discharge burnup.It is worth noting that the radioactive capture reaction of 28Si significantly contributes to the keff uncer-tainty owing to its specific fuel design.However,the keff uncertainty during the full lifetime depletion process was relatively stable,only increasing by 1.12％owing to the low discharge burnup design of small-sized HTGRs.These numerical results are beneficial for neutronics design and core parameters optimization in further uncertainty prop-agation and quantification study for small-sized HTGR.