[Optimization of CD19 chimeric antigen receptor T cell establishment and observation of the killing effect in vitro and in vivo].

Objective: To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Method: Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. Results: The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % vs (47.00±13.46) % , P<0.001; bone marrow: (1.82±0.29) % vs (37.30±1.44) % , P<0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. Conclusion: This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.

Strength-fracture toughness synergy strategy in ostrich tibia's compact bone: Hierarchical and gradient.

Ostriches are the fastest bipeds in the world, but their tibias are very thin. How the thin tibia can withstand the huge momentum impacts of the heavy body during running? The present work revealed that the combination of hierarchical and gradient design strategies was the main reason for their high strength and fracture toughness. The microstructure of ostrich's tibias compact bone was self-assembled into the 6-level hierarchical structure from the hydroxyapatite (HAP) crystals, collagen fiber (sub-nano), mineralized collagen fiber (nano-), mineralized collagen fiber bundle (sub-micro), lamellae (micro-) and osteon (macro-scales). The most distinctive design in the ostrich compact bone was that the HAP crystals were embedded in collagen fibers as well as wrapped in the outer layer of mineral collagen fibers (MCFs) in the form of HAP nanocrystals, thus achieving a high degree of soft and hard combination from the nanoscale. The bending strength was gradient-structure dependent and up to 787.2 ± 40.5 MPa, 4 times that of a human's compact bone. The fracture toughness (KJc) is 5.8 ± 0.1 MPa m1/2. Several toughening mechanisms, such as crack deflection/twist, bridging, HAP fibers pulling-out, and fracture of the MCF bundles were found in the compact bone.

Integrated proteome and lipidome analysis of naturally aged safflower seeds varying in vitality.

Seed ageing has an important effect on germination and productivity. During natural ageing, seed vigour decreases rapidly but, to date, the molecular mechanisms underlying this decrease have not been fully elucidated. Using omics, some of the details regarding seed vigour decline during natural ageing might be elucidated through integrated analysis. Safflower seed germination and physio-biochemical changes during natural ageing (stored for 4, 16 and 28 months) were determined. Proteome and lipidome profiling during natural seed ageing was performed, and the differentially expressed proteins and lipid metabolite species analysed. The surface and internal structures of cotyledons were observed. An integrating analysis of the proteome and lipidome was also carried out. Natural seed ageing significantly decreased safflower seed germination and vigour. 4,184 proteins and 1,193 lipids were quantified, both of which show huge differences among the different naturally aged seeds. The surface of the cotyledons collapsed and cracked, and the oil bodies become looser during natural ageing. The total content of DAG and PA increased, while the content of TAG and PL (PC, PE, PS, PI and PL) significantly decreased during seeds ageing. Two lipase genes (HH-026818-RA and HH-025320) likely participated in this degradation of lipids. We conclude that the enzymes that participate in glycerolipid metabolism and fatty acid degradation probably lead to the degradation of oil bodies (TAG) and membrane lipids (PC, PE, PS, PI, PG) and, ultimately, destroy the structure, causing a decline in seed vigour during natural seed ageing.

Machine Learning Reveals the Seismic Signature of Eruptive Behavior at Piton de la Fournaise Volcano.

Volcanic tremor is key to our understanding of active magmatic systems, but due to its complexity, there is still a debate concerning its origins and how it can be used to characterize eruptive dynamics. In this study we leverage machine learning techniques using 6 years of continuous seismic data from the Piton de la Fournaise volcano (La Réunion island) to describe specific patterns of seismic signals recorded during eruptions. These results unveil what we interpret as signals associated with various eruptive dynamics of the volcano, including the effusion of a large volume of lava during the August-October 2015 eruption as well as the closing of the eruptive vent during the September-November 2018 eruption. The machine learning workflow we describe can easily be applied to other active volcanoes, potentially leading to an enhanced understanding of the temporal and spatial evolution of volcanic eruptions.

[Clinical application of TCGA molecular classification in endometrial endometrioid carcinoma].

Objective: To explore molecular characteristics of endometrial endometrioid cancer according to The Cancer Genome Atlas (TCGA) based molecular classification of endometrial carcinomas and to confirm simple and clinically applicable surrogate methodologies in pathological practice. Methods: Two hundred and twenty-eight cases of endometrial endometroid adenocarcinomas (EnACs) collected from August 2001 to August 2017 from Peking University Health Science Center, Peking University Third Hospital were molecularly categorized by using Sanger sequencing for the exonuclease domain mutations (EDM) of POLE, and by immunohistochemistry for p53 and mismatch repair (MMR) proteins. The cohort was classified into polymerase-E exonuclease domain mutation (POLE EDM), mismatch repair deficiency (MMR-D), p53 abnormal (p53-abn) and p53 wild type (p53-wt) groups. The correlation between molecular subgroups and the clinical-pathological features including prognosis were analyzed. Results: The cohort was distributed as follows: 11(4.8%) POLE EDM, 47(20.6%) MMR-D, 9(4.0%) p53-abn and 161(70.6%) p53-wt. p53-wt subgroup patients demonstrated significantly higher lymph node metastasis (P=0.011) and more advanced stage (P=0.036) than those of somatic hypermutation group cases (POLE EDM and MMR-D). In the FIGO grade 2-3 EnACs cohort, TCGA molecular subtyping was significantly correlated with progression-free survival and overall survival (P=0.043). POLE EDM subgroup had the best survival, while p53-abn subgroup had the worst. Conclusions: Identification of POLE EDM and MMR-D subgroups provides independent and highly valuable prognostic information beyond established histological classification. Based on immunohistochemistry of MMR, p53 and POLE mutational analysis, this pragmatic molecular classification scheme can be served as a reliable surrogate for TCGA molecular classification, which has potential to be used routinely in Chinese pathological practice.

In vitro degradation and mechanical integrity of Mg-Zn-Ca alloy coated with Ca-deficient hydroxyapatite by the pulse electrodeposition process.

The key to manufacturing magnesium-based alloys that are suitable as biodegradable orthopaedic implants is how to adjust their degradation rates and mechanical integrity in the physiological environment. In this study, to solve this challenge, a soluble Ca-deficient hydroxyapatite (Ca-def HA) coating was deposited on an Mg-Zn-Ca alloy substrate by pulse eletrodeposition. This deposition can be demonstrated by X-ray diffractometry and energy dispersion spectroscopy analyses, and the Ca/P atomic ratio of as-deposited coating is about 1.33 (within the range from 1.33 to 1.65). By regulating the appropriate pulse amplitude and width, the Ca-def HA coating shows better adhesion to Mg-Zn-Ca alloy, whose lap shear strength is increased to 41.8+/-2.7 MPa. Potentiodynamic polarization results in Kokubo's simulated body fluid (SBF) indicate that the corrosion potential of Mg alloy increases from -1645 to -1414 mV, while the corrosion current density decreases from 110 to 25 microA/cm(2), which illustrates that the Ca-def HA coating improves the substrate corrosion resistance significantly. Since orthopaedic implants generally serve under conditions of stress corrosion, the mechanical integrity of the Mg-Zn-Ca alloy was measured using the slow strain rate tensile (SSRT) testing technique in SBF. The SSRT results show that the ultimate tensile strength and time of fracture for the coated Mg-Zn-Ca alloy are higher than those of the uncoated one, which is beneficial in supporting fractured bone for a longer time. Thus Mg-Zn-Ca alloy coated with Ca-def HA is be a promising candidate for biodegradable orthopaedic implants, and is worthwhile to further investigate the in vivo degradation behavior.