To explore the therapeutic effect of abiterone combined with dexamethasone by reporting a case of metastatic castration-resistant prostate cancer (mCRPC) who was treated with abiterone combined with prednisone and then changed to abiterone combined with dexamethasone. A 61-year-old man was admitted to Sir Run Run Shaw Hospital in November 2017 due to elevated prostate-specific antigen (PSA) at 11 ng/ml. Prostate MRI showed that abnormal signal intensities in the periphery of the prostate which was considered as prostate cancer. Consideration of metastases in the right iliac crest; partial signal changes in the medial seminal vesicles of both sides which were considered involved. The prostate needle biopsy demonstrated that left prostate adenocarcinoma while Gleason score: 5+ 4=9, diagnosed as metastatic hormone-sensitive prostate cancer (mHSPC). In December 2017, the patient underwent robot assisted laparoscopic radical prostatectomy. Prostatic adenocarcinoma was confirmed by postoperative pathology, Gleason score: 5+ 4=9. The bilateral seminal vesicles and nerves were invaded. The level of PSA was monitored during the continuous postoperative treatment using bicalutamide and goserelin.The PSA was 0.32, 0.15, and 1.72 ng/ml in February, June and October of 2018. In October 2018, the testosterone was 40 ng/dl, considering the biochemical progress. In November 2018, the PSA was 2.51ng/ml, considering entering mCRPC. The treatment plan was switched to abiraterone, prednisone and goserelin, and PSA was monitored. In January, March, and June of 2019, the PSA was 0.1, 0.03, and 1.1 ng/ml. By March 2020, it had reached 8.9 ng/ml. The treatment plan was changed to abiraterone and dexamethasone. In April, July and September of 2020, PSA was 7.9, 3.98, and 2.58 ng/ml respectively. The treatment is still ongoing.Abiraterone combined with prednisone is still effective after asymptomatic PSA progression in mCRPC.

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.
Animals ; Animals, Wild ; Bias (Epidemiology) ; China* ; Codon, Initiator ; Codon, Terminator ; DNA, Intergenic ; Genes, rRNA ; Genetic Variation ; Genome ; Genome, Mitochondrial* ; Mammals ; Phylogeny ; RNA, Transfer ; Sequence Analysis* ; Tigers* ; Toxascaris*

Objective:To establish a fluorescent reporter human induced pluripotent stem cell line (hiPSCs) for monitoring the expression of visual system homeobox 2 ( VSX2). Methods:VSX2_small guide RNA (sgRNA) was inserted into vector PX459 to construct knockout plasmid, and the P2A-eGFP knock-in donor plasmid was conducted at the same time.The two plasmids were transfected into BC1-hiPSCs.Single cell clones were generated after treatment of puromycin.Correct insertion was confirmed by PCR and Sanger sequencing.The isogenicity of the parental and the reporter hiPSCs was confirmed by STR analysis and karyotyping.Pluripotency capacity of the reporter hiPSCs was analysed by reverse trascription PCR and immunofluorescence.Three-germ-layer formation experiment was carried out to analyse the multi-lineage differentiation ability of the reporter hiPSCs.The reporter hiPSCs were further differentiated to obtain three-dimension (3D) retinal organoids, and immunofluorescence was used to identify the co-localization of the enhanced green fluorescent protein (eGFP) and VSX2.Results:A VSX2 eGFP reporter hiPSC clone was successfully obtained by CRISPR/Cas9 technology, which was consistent with the parental hiPSCs (BC1-hiPSCs) in morphology, without any chromosomal aberrations or cell line cross-contamination.Reverse transcription PCR assay and immunofluorescence showed obvious positive expressions of iPSCs markers in BC1- VSX2 eGFP-iPSCs, including NANOG, OCT4, SOX2, DNMT3B and GDF3 mRNA as well as NANOG, OCT4, SSEA4 and TRA-1-60 protein.The α-fetoprotein (AFP), α-smooth muscle actin (α-SMA) and neuronal class Ⅲ β-tubulin (TUJ1) were expressed in endoderm, mesoderm and ectoderm, respeetively, derived from BC1- VSX2 eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1- VSX2 eGFP-iPSCs by immunofluorescence. Conclusions:VSX2 fluorescent reporter hiPSCs is successfully generated, which can monitor the temporal and spatial expression changes of VSX2 protein in real time, providing a powerful tool for evaluation of retina development mechanism and cell therapy.

Cannabis is a traditional industrial crop that has been used for thousands of years for medicines， foods， and textiles. Due to its active components， cannabis has attracted extensive attention in the pharmaceutical industry at home and abroad. Currently， 55 countries around the world have legalized medical marijuana， and two provinces in China， Yunnan and Heilongjiang， can legally cultivate and process industrial hemp. However， the low content of cannabidiol （CBD） in industrial hemp is not conducive to subsequent development and research. Based on this， the author took medicinal cannabis， defined by CHEN Shilin′s team as tetrahydrocannabinol （THC） content<0.3% and high CBD content， as the research object， and reviewed four aspects of the active ingredients of medicinal cannabis， the isolation and purification technology of CBD， the development and application of cannabinoid-related products and the breeding methods of medicinal cannabis. Through combing， it is suggested that subsequent research should focus on excavation of genes of CBD synthesis， molecular breeding of evolutionary cannabis by gene editing technology， development of green extraction process， discovery of more active ingredients， and high yield of CBD through synthetic biology and cell-free system， with a view to provide reference for the development and application of medicinal cannabis in China.

Objective: To prepare human peripheral blood T lymphocytes with TIM3 (T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system. The transfection efficiency was examined 24 h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect hTIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of (41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0% (all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenic peptide;and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group (P<0.05). Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ (P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells (P<0.05). Conclusion: It’s simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.

OBJECTIVE： To establish a method for simultaneous determination of α-pinene， β-pinene， limonene and α-terpineol in volatile oil of Forsythia suspensa. METHODS： GC method was adopted. The determination was performed on HP-5 capillary column through temperature-programmed route. The inlet temperature was 230 ℃， and detector temperature was 250 ℃； split sampling was applied （split ratio of 8 ∶ 1）； the air flow rate was 300 mL/min， the hydrogen flow rate was 30   mL/min， the tail gas flow rate was 30 mL/min， and the injection volume was 1 μL. Using limonene as internal reference， relative correction factors of α-pinene， β-pinene and α-terpineol were established， and the reproducibility of relative correction factors were investigated by using different chromatographs and columns， and chromatographic peak location of components was measured. The contents of above components were calculated with QAMS， and then compared with the results of external standard method. RESULTS： The linear range of α-pinene， β-pinene， limonene and α-terpineol were 16.5-990.0， 38.1-2 287.5， 8.2-491.2， 2.4-142.5  μg/mL， respectively （r≥0.999 1）. RSDs of precision， reproducibility and stability tests were all lower than 3％ （n＝6）. Average recoveries were 99.7％-105.5％（RSD＜4％，n＝9）. Compared with limonene （1.00），the average relative correction factors of α-pinene， β-pinene and α-terpineol were 0.91，0.86 and 1.11（n＝3）； relative retention time were 0.69-0.74， 0.81-0.86， 1.25-1.35（RSD＜3％，n＝3）. By using different chromatographs and columns， RSDs of relative correction factors were 0.21％-4.65％（n＝6）. Compared with external standard method， determination results of above 4 components were consistent （the absolute value of relative error were all less than 7％）. CONCLUSIONS： QAMS can be used for simultaneous determination of 4 kinds of effective components in volatile oil from F. suspensa.