Medical and pharmaceutical research laboratories encompass a wide range of study areas.They utilize diverse materials ranging from animals and microorganisms to nanoparticles and other substances.However,as laboratory waste increases,more biosafety risks are created.In this context,we outlined the safety risks associated with gene amplification,gene recombination,research involving pathogenic microorganisms,nanotechnology,animal experiments,genetically modified animals,and experimental waste.Additionally,we here in propose preventive measures to mitigate laboratory biosafety risks.These measures primarily involve the development of strict legal frameworks,improvement of hardware infrastructure,strengthening of safety awareness,and enhancement of education and training programs.

Objective To observe the functional recovery situation after prostate cancer radical opera-tion with maximal preservation of the periprostatic structures (MS-RARP).Methods Sixty-seven patients with localized prostatic cancer undergoing MS-RARP in the Second Affiliated Hospital of Army Military Med-ical University from June 2019 to June 2022 were selected as the study subjects.The changes of urinary con-trol function,erection function,quality of life and total prostate specific antigen (t-PSA) were compared at the time between before operation and postoperative 6 weeks,3 months,6 months,12 months.The expanded pros-tate cancer index composite index (EPIC-50) was used to evaluate the urinary control function,the interna-tional index of erectile function (IIEF-5) questionnaire was used to evaluate the erection function of the pa-tients,and EPIC-50 and the american urological associated symptom score rating scale (AUA-SS) were used to evaluate the life satisfaction of the patients.Results The urinary control function recovery rate,erection function recovery rate and life quality satisfaction rate in postoperative 6 weeks,3 months,6 months,12 months were 71.8％,95.5％,97.3％ and 98.1％;31.7％,38.1％,41.3％ and 44.4％;38.9％,83.6％,88.1％ and 97.0％,respectively.The median t-PSA was gradually decreased,which were 0.026(0.010,0.410),0.009 (0.003,0.060),0.006(0.001,0.050)and 0.004(0.001,0.006)ng/mL,respectively.The life quality satisfac-tion rates at various postoperative time points were significantly increased when compared with before opera-tion (P<0.05).t-PSA was significantly decreased compared with before operation (P<0.05).Conclusion RARP could protect the postoperative urinary function and sexual function,and increase the postoperative life quality of the patients.

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Humans ; Induced Pluripotent Stem Cells ; Sirolimus/metabolism* ; Caspase 9/metabolism* ; RNA, Guide, CRISPR-Cas Systems ; Pluripotent Stem Cells/metabolism* ; Cell Differentiation ; Puromycin/metabolism*

Post-amputation pain causes great suffering to amputees, but still no effective drugs are available due to its elusive mechanisms. Our previous clinical studies found that surgical removal or radiofrequency treatment of the neuroma at the axotomized nerve stump effectively relieves the phantom pain afflicting patients after amputation. This indicated an essential role of the residual nerve stump in the formation of chronic post-amputation pain (CPAP). However, the molecular mechanism by which the residual nerve stump or neuroma is involved and regulates CPAP is still a mystery. In this study, we found that nociceptors expressed the mechanosensitive ion channel TMEM63A and macrophages infiltrated into the dorsal root ganglion (DRG) neurons worked synergistically to promote CPAP. Histology and qRT-PCR showed that TMEM63A was mainly expressed in mechanical pain-producing non-peptidergic nociceptors in the DRG, and the expression of TMEM63A increased significantly both in the neuroma from amputated patients and the DRG in a mouse model of tibial nerve transfer (TNT). Behavioral tests showed that the mechanical, heat, and cold sensitivity were not affected in the Tmem63a^-/- mice in the naïve state, suggesting the basal pain was not affected. In the inflammatory and post-amputation state, the mechanical allodynia but not the heat hyperalgesia or cold allodynia was significantly decreased in Tmem63a^-/- mice. Further study showed that there was severe neuronal injury and macrophage infiltration in the DRG, tibial nerve, residual stump, and the neuroma-like structure of the TNT mouse model, Consistent with this, expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β all increased dramatically in the DRG. Interestingly, the deletion of Tmem63a significantly reduced the macrophage infiltration in the DRG but not in the tibial nerve stump. Furthermore, the ablation of macrophages significantly reduced both the expression of Tmem63a and the mechanical allodynia in the TNT mouse model, indicating an interaction between nociceptors and macrophages, and that these two factors gang up together to regulate the formation of CPAP. This provides a new insight into the mechanisms underlying CPAP and potential drug targets its treatment.
Animals ; Mice ; Amputation, Surgical ; Chronic Pain/pathology* ; Disease Models, Animal ; Ganglia, Spinal/pathology* ; Hyperalgesia/etiology* ; Ion Channels/metabolism* ; Macrophages ; Neuroma/pathology*

ObjectiveTo explore the possible mechanism of reducing cholecystitis and preventing cholelithiasis by Dahuang Lingxian Formula（大黄灵仙方， DLF）. MethodsFifty SD rats were randomly divided into blank group， model group， DLF group， DLF + blank inhibitor group， and DLF + inhibitor group， with 10 rats in each group. The rat model of cholecystitis was established by lipopolysaccharide （LPS） induction in all the groups except for the blank group. Rats in DLF group， DLF + blank inhibitor group and DLF + inhibitor group received intragastric administration of 320 mg/ （kg·d） of DLF 3 days before the preparation of cholecystitis model， while those in blank group and model group were given 2 ml/100 g of distilled water by gastric， twice a day， for 6 consecutive days. Hematoxylin-eosin （HE） staining was used to observe the histopathologic changes of bile duct tissues in each group. The expression of nuclear factor κB （NF-κB） protein in bile duct tissues was detected by immunohistochemistry. The expression levels of interleukin1β （IL-1β） and tumor necrosis factor α （TNF-α） in serum of rats were detected by enzym-linked immunosorbent assay （ELISA）. The mRNA expression levels of miRNA-30b， transforming growth factor β1 （TGF-β1）， nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） and bone morphogenetic protein 2 （BMP2） in bile duct tissues were detected by real-time PCR， and the protein expression levels of TGF-β1， NLRP3 and BMP2 were detected by Western blot. ResultsCompared to those in the blank group， the structure of the bile duct in the model group was abnormal， and a large number of lymphocytes， plasma cell infiltration and bile canaliculi dilation were seen in the portal area； the positive expression of NF-κB protein increased； there was nuclear infiltration； the expressions of serum inflammatory factors IL-1β and TNF-α， as well as the mRNA and protein expressions of TGF-β1， NLRP3 and BMP2 in bile duct tissue significantly increased， while the expression level of miRNA-30b significantly decreased （P＜0.01）. Compared to those in the model group， the pathological morphology of the bile ducts in the DLF group and DLF + blank inhibitor group was improved， and the infiltration of inflammatory cells was reduced， with decreased positive expression of NF-κB and nuclear infiltration； expression levels of serum inflammatory factors IL-1β and TNF-α， and the mRNA and protein expressions of TGF-β1， NLRP3 and BMP2 in bile duct tissue decreased， while the expression level of miRNA-30b significantly increased （P＜0.05 or P＜0.01）. Compared to the model group， those indicators in the DLF + inhibitor group was not significantly improved （P＞0.05）. There was no significant difference in the indicators between the DLF group and the DLF + blank inhibitor group （P＞0.05）. ConclusionDLF may play a role in delaying the progression of cholelithiasis by regulating the expression of miRNA-30b and inflammation-fibrosis related factors.

Stress, Mechanical ; Biomechanical Phenomena ; Head ; Brain

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.
Animals ; Animals, Genetically Modified ; Gene Knockout Techniques ; HLA Antigens ; Humans ; Nuclear Transfer Techniques ; Swine ; Transplantation, Heterologous

Objective To explore the effect of platelet-rich plasma (PRP) on the generation of reactive oxygen species (ROS) and the phenotypes of photo-aging fibroblasts.Methods A photoaging cell model by repeating UVB irradiation was treated using appropriate concentration of PRP;Cell morphology and the rate of aging dying were observed under inverted microscope 24 hours later after establishment of the cell model;The expression of ROS between experimental and control group was detected using fluorescence microscope after single UVB irradiation.The relative intensity of fluorescence was analyzed using flow cytometry.Results PRP could ameliorate the large and sprawl appearance of photoaging fibroblasts obviously,reduce the generation of ROS as well as decrease the relative intensity of ROS.Conclusions PRP can decrease the level of intracellular oxidative stress caused by UVB irradiation,reduce the generation of ROS and ameliorate the senescence-like phenotypes of pho toaging fibroblasts.

The voltage-gated Na channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Biotin ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Female ; Ganglia, Spinal ; cytology ; HEK293 Cells ; Humans ; Hybridomas ; chemistry ; Hyperalgesia ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; immunology ; metabolism ; Neuralgia ; drug therapy ; metabolism ; Protein Binding ; drug effects ; Recombinant Proteins ; biosynthesis ; therapeutic use ; Sensory Receptor Cells ; drug effects ; physiology

Based on the necessity of carrying out bioethics education to the probationer nurses, this paper put forward to introduce psychological sitcom to the probationer nurses′ bioethics education and described the practice process in detail. It also discussed the mechanism to improve the effectiveness of bioethics education from the per-spectives of creation, dramatization, and performance of psychological sitcom.