Unexpected mutations occurred in CRISPR/Cas9 edited Drosophila analyzed by deeply whole genomic sequencing.

CRISPR/Cas9 possesses the most promising prospects as a gene-editing tool in post-genomic researches. It becomes an epoch-marking technique for the features of speed and convenience of genomic modification. However, it is still unclear whether CRISPR/Cas9 gene editing can cause irreversible damage to the genome. In this study, we successfully knocked out the WHITE gene in Drosophila, which governs eye color, utilizing CRISPR/Cas9 technology. Subsequently, we conducted high-throughput sequencing to assess the impact of this editing process on the stability of the entire genomic profile. The results revealed the presence of numerous unexpected mutations in the Drosophila genome, including 630 SNVs (Single Nucleotide Variants), 525 Indels (Insertion and Deletion) and 425 MSIs (microsatellite instability). Although the KO (knockout) specifically occurred on chromosome X, the majority of mutations were observed on chromosome 3, indicating that this effect is genome-wide and associated with the spatial structure between chromosomes, rather than being solely limited to the location of the KO gene. It is worth noting that most of the mutations occurred in the intergenic and intron regions, without exerting any significant on the function or healthy of the animal. In addition, the mutations downstream of the knockout gene well beyond the upstream. This study has found that gene editing can lead to unexpected mutations in the genome, but most of these mutations are harmless. This research has deepened our understanding of CRISPR/Cas9 and broadened its application prospects.

GB7 acetate,a galbulimima alkaloid from Galbulimima belgraveana,possesses anticancer effects in colorectal cancer cells / 药物分析学报

GB7 acetate is a galbulimima alkaloid obtained from Galbulimima belgraveana.However,information regarding its structure,biological activities,and related mechanisms is not entirely available.A series of spectroscopic analyses,structural degradation,interconversion,and crystallography were performed to identify the structure of GB7 acetate.The MTT assay was applied to measure cell proliferation on human colorectal cancer HCT 116 cells.The expressions of the related proteins were measured by Western blotting.Transmission electron microscopy(TEM),acridine orange(AO)and monodansylcadaverine(MDC)staining were used to detect the presence of autophagic vesicles and autolysosomes.A transwell assay was performed to demonstrate metastatic capabilities.Oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)assays were performed to determine the mitochondrial oxidative phosphorylation(OXPHOS)and glycolysis activity of HCT 116 cells.The data showed that GB7 acetate suppressed the proliferation and colony-forming ability of HCT 116 cells.Pretreatment with GB7 acetate significantly induced the formation of autophagic vesicles and autolysosomes.GB7 acetate upregulated the expressions of LC3 and Thr172 phosphorylated adenosine 5'-monophosphate(AMP)-activated pro-tein kinase α(p-AMPKα),which are key elements of autophagy.In addition,GB7 acetate suppressed the metastatic capabilities of HCT 116 cells.Additionally,the production of matrix metallo-proteinase-2(MMP-2)and MMP-9 was reduced,whereas the expression of E-cadherin(E-cad)was upregulated.Furthermore,GB7 acetate significantly reduced mitochondrial OXPHOS and glycolysis.In conclusion,the structure of the novel Galbulimima alkaloid GB7 acetate was identified.GB7 acetate was shown to have anti-proliferative,pro-autophagic,anti-metastatic,and anti-metabolite capabilities in HCT 116 cells.This study might provide new insights into cancer treatment efficacy and cancer chemoprevention.

Implementation status and suggestions of centralized volume-based procurement policy for drugs and medical consumables in China / 中国药房

OBJECTIVE To provid e reference and sugge stions for improving the volume-based procurement of drugs and medical consumables （hereinafter referred to as “consumables”）in China. METHODS The relevant policy documents of centralized volume-based procurement of drugs and consumables published from November 2018 to November 2021 were retrieved ； the implementation status and problems of centralized volume-based procurement of drugs and consumables in China were analyzed by using the policy analysis method and referring to relevant research literatures. RESULTS & CONCLUSIONS National health department and healthcare security administration guaranted the rational use of selected products in medical institutions through incentive and supervision measures ；healthcare security administration should optimize the way of medical insurance payment ， promote the medical institutions to control the fees by themselves ，and conduct the credit evaluation of bidding and procurement ； medical products administration should evaluate the consistency of drugs and supervise the quality of selected products. With the normalization of centralized volume-based procurement of drugs and consumables organized by the state and trans-regional alliance ， the drug varieties and dosage forms included in centralized procurement were increasingly in line with the demand of Chinese pharmaceutical market. The price of most selected drugs decreased by more than 50％，and the decrease of consumables was significantly higher than that of drugs. The selected enterprises were mainly domestic generic drug enterprises ，and domestic consumables had gradually become the competitors and substitute of imported consumables. However ，there were still some problems such as repeated bidding and procurement in various alliances and provinces （autonomous regions and municipalities ）， unclear construction of compensation mechanism in medical institutions ，inconsistent bidding and procurement rules and quality evaluation standards for consumables ，low localization rate of some consumables ，low innovation level and profitability of pharmaceutical enterprises and consumables manufacturers. Local centralized volume-based procurement should be encouraged ，and the bidding and procurement rules and quality evaluation standards of “one product ，one policy ”should be gradually established. Great importance should be paid to the construction of compensation mechanism of medical institutions ，standardize zhangqiuyu739632@126.com the dynamic adjustment of medical serv ice prices ；pharma- ceutical enterprises and consumables manufacturers should increase research and development investment to transform into innovative and diversified enterprises ，so as to improve the competitiveness of domestic drugs and consumables.

Pathogenic mechanisms of Anaplasma phagocytophilum / 中华微生物学和免疫学杂志

Anaplasma phagocytophilum ( A. phagocytophilum) is tick-borne obligate pathogen that parasitizes rodents, ruminants, deer, horses and human. Ticks can transmit A. phagocytophilum to human resulting in a disease called anaplasmosis. With the increase in outdoor activities, the chances of exposing to ticks increase in human and the probability of suffering from anaplasmosis increases correspondingly. Most patients can recover from anaplasmosis after doxycycline therapy, but immunocompromised patients are at risk of seizure, renal failure and even death. A comprehensive understanding of A. phagocytophilum pathogenic mechanisms can provide new therapeutic strategies for the treatment of critically ill patients. Therefore, this review first gave examples of pathogenesis-related proteins of A. phagocytophilum, and then summarized the current research status of the pathogenic mechanism of this pathogen from three aspects of interference with cytoskeletal remodeling, inhibition of host cell apoptosis and dysfunction of the innate immune response, and proposed main issues to be addressed.

Preparation of warangalone-loaded liposomes and its inhibitory effect on breast cancer cells / 南方医科大学学报

OBJECTIVE@#To prepare warangalone-loaded thermosensitive liposomes (WLTSL) and evaluate its inhibitory effect on breast cancer cells .@*METHODS@#MTT assay was used to assess the changes in proliferation of 3 breast cancer cell lines (MDA-MB-231, MCF7, and SKBR3) following treatment with warangalone, soy isoflavone and genistein. Colony-forming assay and wound healing assay was used to assess colony forming activity and migration of MDA-MB-231 cells treated with warangalone. The effect of warangalone on the expression of MMP2 and MMP9 in MDA-MB-231 cells was examined with Western blotting. The thermosensitive liposomes (TSL) and WLTSL were prepared using a thin film hydration method, and the morphology, size, encapsulation efficiency and stability of the prepared liposomes were characterized using transmission electron microscopy, dynamic light scattering scanning and UV spectrophotometry. MTT assay was used to examine the inhibitory effect of WLTSL on mouse breast cancer cells (4T1) .@*RESULTS@#Warangalone showed stronger anti-proliferation effects than soy isoflavones and genistein in the 3 human breast cancer cell lines and significantly inhibited colony formation by MDA-MB-231 cells. Treatment with warangalone significantly inhibited migration of the breast cancer cells and down-regulated the cellular expressions of MMP2 and MMP9. The prepared TSL and WLTSL presented with a homogeneous, irregular spherical morphology, with a mean particle size of 56.23±0.61 nm, a polymer dispersity index of 0.241±0.014, a Zeta potential of -40.40±0.46 mV, and an encapsulation efficiency was 87.68±2.41%. WLTSL showed a good stability at 4 ℃ and 37 ℃ and a stronger inhibitory effect than warangalone in 4T1 cells.@*CONCLUSIONS@#Warangalone inhibits the proliferation, migration and invasion of breast cancer cells, and the prepared WLTSL possesses good physical properties and strong anti-breast cancer activity.

Mechanism of Treg down-regulation by immunization with attenuated activated autologous T cells / 上海交通大学学报（医学版）

Objective To explore the mechanism of down-regulation of regulatory T cells (Treg) by immunization with attenuated activated autologous T cells. Methods Aulologous T cells were activated with ConA in vitro. Mice were immunized subcutaneously and inlraperitoneally every 5 days for 3 times (5 ×10~6 per time for each mouse), and the number and function of Treg were examined. PBS was subcutaneously injected for control group. Serum level of anti-mouse CD25 antibody was measured by ELISA. The number and function of Treg was detected by serum adoptive transfer and proliferation and inhibition assays. Results Compared with control group, there were less CD4~+ CD25~+ Foxp3~+ Treg in the mice after immunization (P < 0. 01), the immunosuppression ability decreased (P<0. 01), and the level of anti-CD25 antibody increased (P <0.01). Adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice (P<0. 01). Conclusion Immunization with attenuated activated autologous T cells induces more anti-CD25 antibody, which may further down-regulate CD4~+CD25~+Foxp3~+ Treg expansion and function in vivo.