The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

Objective:To evaluate the role of hippocampal activating transcription factor 5 (ATF5) in cognitive impairment induced by neuropathic pain and the relationship with mitochondrial unfolded protein response(mtUPR) in mice.Methods:This study was conducted in 2 parts. Experiment Ⅰ Twenty-four SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups ( n=12 each) using a random number table method: sham operation group (S1 group) and neuropathic pain group (NP group). Neuropathic pain was induced by chronic constriction injury to the sciatic nerve. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before developing the model and at 7, 14, 21 and 28 days after developing the model. Mouse cognitive function was assessed using the novel object recognition test from 30-31 days after developing the model. After the end of the novel object recognition test, mice were sacrificed and the hippocampal CA1 region was harvested for determination of the expression of ATF5 (by Western blot) and the expression of ATF5 in neurons, microglia and astrocytes (by immunofluorescence double staining). Experiment Ⅱ Thirty-six SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (S2 group), neuropathic pain + ATF5 up-regulation group (NA group), and neuropathic pain + empty virus group (NE group). On day 14 after developing the model, a virus that specifically up-regulated ATF5 expression in neurons and empty virus were injected into the hippocampal CA1 region. The MWT and TWL were measured at days 28 and 35 after developing the model. The novel object recognition test was performed on day 36 after developing the model to evaluate the cognitive function. After the end of the behavioral test, mice were sacrificed and the hippocampal CA1 region was harvested for detection of the expression of ATF5 and mtUPR marker proteins (Lon protease [LONP1] and heat shock protein 60 [HSP60]) by Western blot. Results:Experiment Ⅰ Compared with S1 group, no statistically significant change was found in the MWT and TWL before developing the model ( P>0.05), the MWT and TWL were significantly decreased on days 7, 14, 21 and 28 after developing the model, the discrimination index (DI) was decreased at day 31 after developing the model, the expression of ATF5 was down-regulated, the expression of ATF5 in neurons was down-regulated ( P<0.05), and no statistically significant change was found in the expression of ATF5 in mircrolia and astrocytes in NP group ( P>0.05). Experiment Ⅱ Compared with S2 group, the MWT and TWL were significantly decreased on days 28 and 35 after developing the model in NE group and NA group, DI was decreased, and the expression of ATF5, LONP1 and HSP60 was down-regulated in NE group ( P<0.05), and no significant change was found in NA group ( P>0.05). Compared with NE group, no significant change was found in the MWT and TWL in NA group ( P>0.05), DI was significantly increased, and the expression of ATF5, LONP1 and HSP60 was up-regulated in NA group ( P<0.05). Conclusions:Down-regulated ATF5 in the hippocampus is involved in the process of cognitive impairment caused by neuropathic pain, and the mechanism may be related to the inhibition of mtUPR.

Objective:To evaluate the role of hippocampal activating transcription factor 5 (ATF5) in cognitive impairment induced by neuropathic pain and the relationship with mitochondrial unfolded protein response(mtUPR) in mice.Methods:This study was conducted in 2 parts. Experiment Ⅰ Twenty-four SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups ( n=12 each) using a random number table method: sham operation group (S1 group) and neuropathic pain group (NP group). Neuropathic pain was induced by chronic constriction injury to the sciatic nerve. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before developing the model and at 7, 14, 21 and 28 days after developing the model. Mouse cognitive function was assessed using the novel object recognition test from 30-31 days after developing the model. After the end of the novel object recognition test, mice were sacrificed and the hippocampal CA1 region was harvested for determination of the expression of ATF5 (by Western blot) and the expression of ATF5 in neurons, microglia and astrocytes (by immunofluorescence double staining). Experiment Ⅱ Thirty-six SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (S2 group), neuropathic pain + ATF5 up-regulation group (NA group), and neuropathic pain + empty virus group (NE group). On day 14 after developing the model, a virus that specifically up-regulated ATF5 expression in neurons and empty virus were injected into the hippocampal CA1 region. The MWT and TWL were measured at days 28 and 35 after developing the model. The novel object recognition test was performed on day 36 after developing the model to evaluate the cognitive function. After the end of the behavioral test, mice were sacrificed and the hippocampal CA1 region was harvested for detection of the expression of ATF5 and mtUPR marker proteins (Lon protease [LONP1] and heat shock protein 60 [HSP60]) by Western blot. Results:Experiment Ⅰ Compared with S1 group, no statistically significant change was found in the MWT and TWL before developing the model ( P>0.05), the MWT and TWL were significantly decreased on days 7, 14, 21 and 28 after developing the model, the discrimination index (DI) was decreased at day 31 after developing the model, the expression of ATF5 was down-regulated, the expression of ATF5 in neurons was down-regulated ( P<0.05), and no statistically significant change was found in the expression of ATF5 in mircrolia and astrocytes in NP group ( P>0.05). Experiment Ⅱ Compared with S2 group, the MWT and TWL were significantly decreased on days 28 and 35 after developing the model in NE group and NA group, DI was decreased, and the expression of ATF5, LONP1 and HSP60 was down-regulated in NE group ( P<0.05), and no significant change was found in NA group ( P>0.05). Compared with NE group, no significant change was found in the MWT and TWL in NA group ( P>0.05), DI was significantly increased, and the expression of ATF5, LONP1 and HSP60 was up-regulated in NA group ( P<0.05). Conclusions:Down-regulated ATF5 in the hippocampus is involved in the process of cognitive impairment caused by neuropathic pain, and the mechanism may be related to the inhibition of mtUPR.

Objective:This study intends to analyze the differences of indexes between the death group and the survival group of CSA-AKI patients undergoing CRRT before, during, and after heart surgery, and at the beginning and the end of CRRT, and to study the risk factors affecting the death of patients, so as to provide guidance for improving the prognosis.Methods:From January 2020 to October 2022, a total of 101 patients (59 in the mortality group and 42 in the survival group) were admitted to the Department of Cardiac Surgery at the First Affiliated Hospital of Zhengzhou University for cardiac surgery complicated by acute kidney injury necessitating postoperative continuous renal replacement therapy (CRRT). A retrospective analysis of clinical data was conducted to ascertain the risk factors influencing the 30-day mortality rate.Results:The postoperative complications of the two groups showed that the mortality group had a large proportion of MODS and LCOS ( P<0.05). Multivariate regression analysis showed that intraoperative mean arterial depression, high SOFA score at the beginning of CRRT, high APACHE Ⅱ score at the beginning of CRRT, and low platelet at the end of CRRT were independent risk factors for death( P<0.05). ROC curve analysis shows that SOFA score and APACHE Ⅱ score at the beginning of CRRT have predictive value for the 30-day death risk of CSA-AKI patients receiving CRRT. Conclusion:Low intraoperative MAP, high SOFA and APACHE Ⅱ scores at the beginning of CRRT, and low platelets at the end of CRRT were independent risk factors for 30-day mortality risk in patients with CSA-AKI undergoing CRRT.

Objective To investigate the safety and effectiveness of normal saline as a hydrodissection band versus sodium hyaluronate and biopolysaccharide mixture as a modified hydrodissection band in microwave ablation (MWA) for benign thyroid nodules. Methods A total of 196 patients with 245 benign thyroid nodules(maximum diameter ≥20 mm) treated at Shanghai Ruijin Rehabilitation Hospital from August 2018 to February 2022 were enrolled. Patients were divided into control group (n=114): MWA using normal saline as the hydrodissection fluid; modified group (n=131): MWA using sodium hyaluronate and biopolysaccharide mixture as the hydrodissection fluid. A hydrodissection band (about 10 mm wide) was established before MWA. Intraoperative hydrodissection fluid absorption time was recorded. At the 12-month follow-up endpoint, complications were assessed, and changes in nodule volume (volume reduction rate, VRR) were evaluated. Results The complication rate was significantly lower in the modified group (1.9% vs.14.0%, P<0.05). 92.3% of complications in the control group were thermal injury-related, while no thermal injuries occurred in the modified group. Hydrodissection fluid absorption time was significantly prolonged in the modified group [(15.2±1.6) min vs. (11.0±2.3) min, P<0.05].No significant difference was observed in overall treatment efficacy (nodule VVR) between two groups (95.4% vs. 96.5%, P>0.05). Conclusions The sodium hyaluronate and biopolysaccharide mixture demonstrates prolonged retention time and superior thermal insulation as an hydrodissection fluid in MWA. It reduces adhesion risks and complications such as hoarseness, dysphagia, and thermal injury, thereby enhancing procedural safety and postoperative recovery. MWA is an effective treatment for benign thyroid nodules.

The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

Objective:This study intends to analyze the differences of indexes between the death group and the survival group of CSA-AKI patients undergoing CRRT before, during, and after heart surgery, and at the beginning and the end of CRRT, and to study the risk factors affecting the death of patients, so as to provide guidance for improving the prognosis.Methods:From January 2020 to October 2022, a total of 101 patients (59 in the mortality group and 42 in the survival group) were admitted to the Department of Cardiac Surgery at the First Affiliated Hospital of Zhengzhou University for cardiac surgery complicated by acute kidney injury necessitating postoperative continuous renal replacement therapy (CRRT). A retrospective analysis of clinical data was conducted to ascertain the risk factors influencing the 30-day mortality rate.Results:The postoperative complications of the two groups showed that the mortality group had a large proportion of MODS and LCOS ( P<0.05). Multivariate regression analysis showed that intraoperative mean arterial depression, high SOFA score at the beginning of CRRT, high APACHE Ⅱ score at the beginning of CRRT, and low platelet at the end of CRRT were independent risk factors for death( P<0.05). ROC curve analysis shows that SOFA score and APACHE Ⅱ score at the beginning of CRRT have predictive value for the 30-day death risk of CSA-AKI patients receiving CRRT. Conclusion:Low intraoperative MAP, high SOFA and APACHE Ⅱ scores at the beginning of CRRT, and low platelets at the end of CRRT were independent risk factors for 30-day mortality risk in patients with CSA-AKI undergoing CRRT.

Objective:To develop the Postoperative Subdelirium Syndrome Knowledge, Attitude, and Practice Questionnaire for Surgical Nurses and test its reliability and validity.Methods:Based on the theory of knowledge, attitude, and practice, the preliminary Postoperative Subdelirium Syndrome Knowledge, Attitude, and Practice Questionnaire for Surgical Nurses was developed through literature search and analysis, Delphi expert consultation, and pre-survey. From February to April 2022, convenience sampling was used to select 375 surgical nurses from five Class Ⅲ Grade A hospitals and three Class Ⅲ Grade B hospitals in Zhejiang Province for questionnaire item analysis, exploratory factor analysis, and reliability testing.Results:The final Postoperative Subdelirium Syndrome Knowledge, Attitude, and Practice Questionnaire for Surgical Nurses consisted of 33 items in three dimensions of knowledge, attitude, and practice. Exploratory factor analysis extracted three common factors with a cumulative variance contribution rate of 67.016%. The total Cronbach's α coefficient of the questionnaire was 0.931, and the Cronbach's α coefficients for each dimension were 0.972, 0.906, and 0.932, respectively. The overall test-retest reliability coefficient of the questionnaire was 0.999, and the test-retest reliability coefficients for each dimension were 0.997, 0.994, and 0.998. The questionnaire-level content validity index was 0.889, and the item-level content validity index was 0.833 to 1.000.Conclusions:The Postoperative Subdelirium Syndrome Knowledge, Attitude, and Practice Questionnaire for Surgical Nurses has good reliability and validity, and can be used to evaluate the knowledge, attitude, and practice of postoperative subdelirium syndrome among surgical nurses.

ObjectiveTo explore the amelioration of cognitive dysfunction in diabetes mellitus （DM） by Jianpi Qinghua prescription （JPQH） based on type 2 diabetes （T2DM） model rats. MethodFifty healthy male Wistar rats of SPF grade were randomly divided into control group （n=10） and experimental group （n=40）. The rats in the control group were fed conventionally， while those in the experimental group were fed on a high-sugar， high-fat diet for six weeks and administered with streptozotocin （STZ） for the induction of the DM model. The model rats were randomly divided into model group， sitagliptin group （1.2 g·L^-1）， pioglitazone group （0.8 g·L^-1）， and JPQH group （1.3 g·mL^-1）， with 10 rats in each group. After six weeks of drug intervention， the changes in body weight， blood glucose， and other related indexes of each group were recorded. Enzyme-linked immunosorbent assay （ELISA） was performed to detect the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in the peripheral blood and brain. The Morris water maze test was used to evaluate the cognitive function in rats. Hematoxylin-eosin （HE） staining was used to observe the pathological morphology of the hippocampal CA region. The amyloid β-protein 40 （Aβ₄₀） level was detected by immunohistochemistry. The protein expression of t-tau and p-tau in hippocampal neurons of rats was detected by Western blot. ResultCompared with blank group， the body weight of model group was significantly decreased （P<0.05）， blood glucose level was significantly increased （P<0.01）， inflammatory cytokines TNF-α and IL-1β were increased （P<0.05）， learning and spatial ability were significantly decreased （P<0.01）， the arrangement of hippocampal cells was loose and disordered， and the intercellular space was significantly increased. The number of cells decreased significantly， and the expression of Aβ₄₀ increased significantly. and increased t-tau and p-tau protein content in the hippocampus （P<0.01）. Compared with model group， the JPQH group showed reduced blood glucose （P<0.01）， decreased TNF-α and IL-1β levels in the peripheral blood and cerebrospinal fluid （P<0.05）， a downward trend of IL-6 without a statistical difference， improved learning and spatial memory ability （P<0.01）， densely arranged cells in the hippocampal CA1 area， increased cell number， reduced Aβ₄₀ expression， and decreased p-tau protein expression （P<0.05）. ConclusionJPQH can prevent cognitive dysfunction in DM by reducing inflammatory factor levels， decreasing neurotoxicity caused by Aβ₄₀ deposition， and inhibiting hyperphosphorylation of tau protein in DM rats.

Abstract: Cell death is a fundamental biological phenomenon that is essential for the survival and development of organisms. Cell death can be either a spontaneous programmed process by the host or an accidentally triggered process. According to the different signaling pathway activated by various stimulates, programmed cell death exhibits the lytic or non-lytic morphology. For example, apoptosis, a typical non-lytic form of cell death, exhibits cell shrinkage and induces the formation of apoptotic bodies. Pyroptosis mediated by cysteine-containing aspartate-specific protease-1/11 (caspase-1/11) and necroptosis can induce inflammatory reactions and promote cell lysis to release inflammatory cytokines via triggering the pore-forming mechanism of the cell membrane, representing a typical modes of lytic cell death. In addition, the release of reactive oxygen species caused by the damaged mitochondria may further trigger ferroptosis during the pathogen infection. Programmed cell death can play an immune defensive role by eliminating infected cells and intracellular pathogens and stimulating the innate immune response through the resulting cell corpses. Here, we discuss the molecular mechanisms of five programmed cell death pathways: apoptosis, pyroptosis, ferroptosis, necroptosis and PANoptosis. We describe their roles in the innate immune defense against bacterial infections and give a brief statement of the interactions between the different programmed cell death, hoping to provide new insights for in-depth study of the pathogenic mechanisms of infectious diseases.