Objective To explore the relationship between PANoptosis and hepatic ischemia-reperfusion injury (HIRI), and to screen the key genes of PANoptosis in HIRI. Methods PANoptosis-related differentially expressed genes (PDG) were obtained through the Gene Expression Omnibus database and GeneCards database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were used to explore the biological pathways related to PDG. A protein-protein interaction network was constructed. Key genes were selected, and their diagnostic value was assessed and validated in the HIRI mice. Immune cell infiltration analysis was performed based on the cell-type identification by estimating relative subsets of RNA transcripts. Results A total of 16 PDG were identified. GO analysis showed that PDG were closely related to cellular metabolism. KEGG analysis indicated that PDG were mainly enriched in cellular death pathways such as apoptosis and immune-related signaling pathways such as the tumor necrosis factor signaling pathway. GSEA results showed that key genes were mainly enriched in immune-related signaling pathways such as the mitogen-activated protein kinase (MAPK) signaling pathway. Two key genes, DFFB and TNFSF10, were identified with high accuracy in diagnosing HIRI, with areas under the curve of 0.964 and 1.000, respectively. Immune infiltration analysis showed that the control group had more infiltration of resting natural killer cells, M2 macrophages, etc., while the HIRI group had more infiltration of M0 macrophages, neutrophils, and naive B cells. Real-time quantitative polymerase chain reaction results showed that compared with the Sham group, the relative expression of DFFB messenger RNA in liver tissue of HIRI group mice increased, and the relative expression of TNFSF10 messenger RNA decreased. Cibersort analysis showed that the infiltration abundance of naive B cells was positively correlated with DFFB expression (r=0.70, P=0.035), and the infiltration abundance of M2 macrophages was positively correlated with TNFSF10 expression (r=0.68, P=0.045). Conclusions PANoptosis-related genes DFFB and TNFSF10 may be potential biomarkers and therapeutic targets for HIRI.

Objective:To evaluate the role of small ubiquitin-associated modifier (SUMO) E3 ligase (PIAS)-regulated SUMOylation of peroxisome proliferator-activated receptor γ (PPARγ) in the endogenous protective mechanism against endotoxin-induced acute lung injury (ALI) in mice.Methods:Experiment Ⅰ Twenty-four clean-grade wild type male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (C group), ALI group, ALI+ PPARγ inducer TZD group (ALI+ T group) and ALI+ TZD+ SUMOylation inhibitor anacardic acid group (ALI+ T+ A group). Lipopolysaccharide (LPS) 15 mg/kg was injected into the tail vein to develop the ALI model. In ALI+ T+ A group, anacardic acid 5 mg/kg was intraperitoneally injected at 1 h before LPS administration. In ALI+ T group and ALI+ T+ A group, TZD 50 mg/kg was intraperitoneally injected at 30 min before LPS administration. The mice were sacrificed at 12 h after LPS administration, and the lung tissues were obtained to examine the pathological changes which were scored and to determine the wet/dry (W/D) weight ratio, and expression of PIAS1, PIAS2, PIAS3 and PIASy protein and mRNA (by Western blot or polymerase chain reaction). Experiment Ⅱ Mouse alveolar macrophages (MH-S cells) were cultured in vitro and divided into 4 groups ( n=5 each) using a random number table method: control group (C group), LPS group, LPS+ PIAS2 siRNA group (L+ P group) and LPS+ Con siRNA group (L+ C group). Cells were routinely cultured in group C. Cells were stimulated with 10 μg/ml LPS to develop the model of endotoxin challenge. PIAS2 siRNA 50 nmol/L and Con siRNA 50 nmol/L were transfected at 48 h before LPS was added in L+ P group and L+ C group, respectively. The cells were collected at 24 h of incubation with LPS to determine the cell viability, levels of M1 and M2 alveolar macrophages (by flow cytometry), expression of PIAS2 and PPARγ (by Western blot), co-expression of PPARγ-SUMO1 (by immunoprecipitation) and expression of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) mRNA (by polymerase chain reaction). The ratio of M1/M2 was calculated. Results:Experiment Ⅰ Compared with C group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with ALI group, the lung injury scores and W/D ratio were significantly decreased, and the expression of PIAS2 protein and mRNA was up-regulated in ALI+ T group and ALI+ T+ A group ( P<0.05). Compared with ALI+ T group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was down-regulated in ALI+ T+ A group ( P<0.05). There was no significant difference in the expression of PIAS1, PIAS3 and PIASy protein and mRNA in lung tissues among the four groups ( P>0.05). Experiment Ⅱ Compared with C group, the cell viability was significantly decreased, the expression of PPARγ and co-expression of PPARγ-SUMO1 was up-regulated, the levels of M1 and M2 macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was up-regulated, and the expression of IL-10 mRNA was down-regulated in the other three groups, and PIAS2 expression was significantly up-regulated in L group and L+ C group ( P<0.05). Compared with L group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and PPARγ-SUMO1 co-expression were down-regulated, the M1 macrophage level and M1/M2 ratio were increased, TNF-α mRNA expression was up-regulated, and the expression of IL-10 mRNA was down-regulated in L+ P group ( P<0.05), and no significant change was found in the parameters mentioned above in L+ C group ( P>0.05). Compared with L+ C group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and co-expression of PPARγ-SUMO1 were down-regulated, the level of M1 alveolar macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was down-regulated, and the expression of IL-10 mRNA was up-regulated in L+ P group ( P<0.05). Conclusions:PIAS2-regulated SUMOylation of PPARγ is the endogenous protective mechanism against endotoxin-induced ALI in mice, which may be related to inhibition of macrophage polarization into M1 type and alleviation of inflammatory responses.

OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. CONCLUSION By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans ; COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics* ; RNA ; COVID-19 Testing

Objective To evaluate potential protective effects of inhaled budesonide on bronchopulmonary dysplasia (BPD) in premature infants and its impact on the intelligence development at 1 year of age. Methods A total of 82 preterm infants admitted to the neonatal intensive care center from January 2017 to January 2018 were selected as research subjects. The enrolled subjects were divided into a study group (Budesonide) and a control group (saline) by random number table method, with 41 cases in each group. The incidence of BPD, mortality, hospitalization time, time of withdrawal and oxygenation, and complications were compared between the two groups. The patients were regularly followed up to 1 year old after discharge. The physical growth and Gesell intelligence development of the two groups were compared. Results The incidence of BPD in the study group was significantly lower than that in the control group, and the difference was statistically significant (P<0.05). There was no significant difference in mortality between the two groups. The hospitalization time, weaning and oxygenation time, and 1-week re-intubation rate in the study group were significantly lower than those in the control group (P<0.05). There was no significant difference between the two groups in the incidence of adverse reactions and physical growth at 1 year old and Gesell intelligence evaluation. Conclusion Budesonide aerosol inhalation reduced the incidence of BPD in premature infants, shortened hospitalization and weaning time, and there were no near-term and long-term adverse reactions.

Background: Lumbar disc herniation (LDH) is a common cause of radicular pain, but the mechanism is not clear. In this study, we investigated the engagement of toll-like receptor 4 (TLR4) and the nuclear factor-kappa B (NF-κB) in radicular pain and its possible mechanisms. Methods: An LDH model was induced by autologous nucleus pulposus (NP) implantation, which was obtained from coccygeal vertebra, then relocated in the lumbar 4/5 spinal nerve roots of rats. Mechanical and thermal pain behaviors were assessed by using von Frey filaments and hotplate test respectively. The protein level of TLR4 and phosphorylated-p65 (p-p65) was evaluated by western blotting analysis and immunofluorescence staining. Spinal microglia activation was evaluated by immunofluorescence staining of specific relevant markers. The expression of proand anti-inflammatory cytokines in the spinal dorsal horn was measured by enzyme linked immunosorbent assay. Results: Spinal expression of TLR4 and p-NF-κB (p-p65) was significantly increased after NP implantation, lasting up to 14 days. TLR4 was mainly expressed in spinal microglia, but not astrocytes or neurons. TLR4 antagonist TAK242 decreased spinal expression of p-p65. TAK242 or NF-κB inhibitor pyrrolidinedithiocarbamic acid alleviated mechanical and thermal pain behaviors, inhibited spinal microglia activation, moderated spinal inflammatory response manifested by decreasing interleukin (IL)-1β, IL-6, tumor necrosis factor-α expression and increasing IL-10 expression in the spinal dorsal horn. Conclusions The study revealed that TLR4/NF-κB pathway participated in radicular pain by encouraging spinal microglia activation and inflammatory response.

Background: Lumbar disc herniation (LDH) is a common cause of radicular pain, but the mechanism is not clear. In this study, we investigated the engagement of toll-like receptor 4 (TLR4) and the nuclear factor-kappa B (NF-κB) in radicular pain and its possible mechanisms. Methods: An LDH model was induced by autologous nucleus pulposus (NP) implantation, which was obtained from coccygeal vertebra, then relocated in the lumbar 4/5 spinal nerve roots of rats. Mechanical and thermal pain behaviors were assessed by using von Frey filaments and hotplate test respectively. The protein level of TLR4 and phosphorylated-p65 (p-p65) was evaluated by western blotting analysis and immunofluorescence staining. Spinal microglia activation was evaluated by immunofluorescence staining of specific relevant markers. The expression of proand anti-inflammatory cytokines in the spinal dorsal horn was measured by enzyme linked immunosorbent assay. Results: Spinal expression of TLR4 and p-NF-κB (p-p65) was significantly increased after NP implantation, lasting up to 14 days. TLR4 was mainly expressed in spinal microglia, but not astrocytes or neurons. TLR4 antagonist TAK242 decreased spinal expression of p-p65. TAK242 or NF-κB inhibitor pyrrolidinedithiocarbamic acid alleviated mechanical and thermal pain behaviors, inhibited spinal microglia activation, moderated spinal inflammatory response manifested by decreasing interleukin (IL)-1β, IL-6, tumor necrosis factor-α expression and increasing IL-10 expression in the spinal dorsal horn. Conclusions The study revealed that TLR4/NF-κB pathway participated in radicular pain by encouraging spinal microglia activation and inflammatory response.

Objective:To evaluate the effect of intestinal carbapenem-resistant Enterobacteriaceae (CRE) active screening combined with enhanced intervention in the prevention and control of nosocomial infection in patients admitted to the hematological ward.Methods:Patients who were admitted to the Department of Hematology in a tertiary-care general hospital from March 1, 2017 to December 31, 2019 and underwent chemotherapy or immunosuppressive therapy comprised the intervention group. They were screened for intestinal CRE at least thrice. From December 1, 2016 to February 28, 2017, patients who underwent chemotherapy or immunosuppressive therapy without active intestinal CRE screening in the Department of Hematology formed the control group. Both the patient groups were monitored for CRE infection in real time. The χ2 test was used to compare the changes in the CRE infection rate and mortality in high-risk patients before and after the active screening. Results:During the intervention period, the CRE colonization rate of patients was 16.46% (66/401) ; in terms of disease distribution, the colonization rate of acute leukemia was the highest 23.03% (26/113) . Of the 66 colonized patients, 27 (40.9%) patients were identified as positive for CRE at the first screening, 15 (22.7%) were identified at the time of the second screening, and the remaining 24 (36.4%) were identified at the third or subsequent screening; Carbapenem-resistant Klebsiella pneumoniae (CRPK) strains were dominant among the pathogens, accounting for 54.55% (36/66) . During the active screening period, the CRE infection rate (2.49%) and mortality rate (50.00%) of high-risk patients were significantly lower than those of the controls (11.30% and 69.23%, respectively) . The pathogens of 10 CRE infection patients during the intervention period were exactly the same as the previous active screening pathogens, and the coincidence rate was 100%.Conclusion:The CRE colonization rate was the highest in patients with acute leukemia who were admitted in the hematology wards. CRPK is the main pathogen of CRE colonization, infection, and death. Increasing the frequency of screening can significantly raise the positive rate of screening, Active screening can effectively reduce the incidence and subsequent mortality of CRE in high-risk patients admitted in the hematological wards. High coincidence rate between CRE screening positive pathogens and subsequent CRE infection pathogens. Intestinal CRE screening can serve as an indicator of CRE bloodstream infection in patients with hematological diseases as well as provide information for antibiotics therapy.

Objective:To explore the effect of montelukast sodium on the treatment of children with bronchial asthma.Methods:From January 2018 to October 2019, 96 children with bronchial asthma treated in Hangzhou Dajiangdong Hospital were randomly divided into control group and observation group according to random double blind method, with 48 cases in each group.The control group was treated with glucocorticoids and anti-infection, conventional treatment such as phlegm and expectoration, and the observation group was treated with montelukast sodium on the basis of conventional treatment.The inflammatory factors, pulmonary function indicators and clinical symptoms recovery time were compared between the two groups before and after treatment.Results:The cough, wheezing and lung murmur disappearing time in the observation group were (3.84±1.87)d, (2.68±0.75)d, (4.35±1.05)d, respectively, which were shorter than those in the control group[(5.78±2.01)d, (3.97±1.01)d, (7.16±1.34)d], the differences were statistically significant( t=4.740, 6.879, 11.073, all P<0.05). The expressions of interleukin-8, tumor necrosis factor-ɑ and interleukin-6 in the observation group were lower than those in the control group(all P<0.05). The forced expiratory volume in the first second, peak expiratory velocity and forced expiratory volume in the first second/forced vital capacity in the observation group were higher than those in the control group, the differences were statistically significant( t=5.531, 4.155, 8.090, all P<0.05). Conclusion:Montelukast sodium can improve lung function and reduce serum inflammatory response in children with bronchial asthma, which is of great clinical significance.

Radiotherapy is the most common treatment for nasopharyngeal carcinoma, and the radiotherapy technique is essential for the prognosis of nasopharyngeal carcinoma. Due to the complexity of the structure of the intensity-modulated device and the accuracy of the clinical requirements of radiotherapy, it is inevitable that higher requirements will be imposed on the process of intensity-modulated radiotherapy. Currently, gaps exist in the radiotherapy equipment and personnel qualification among radiotherapy units, and thus the homogenization in the radiotherapy remains to be strengthened in China. With the application of radiotherapy information management system, digital medicine and artificial intelligence technologies in the field of radiotherapy, the original process fails to meet the application needs of the new precise radiotherapy technology. Therefore, this process is designed based on the existing radiotherapy procedures for nasopharyngeal carcinoma in combination with the latest developments in the field of radiotherapy, aiming to establish a novel standard process recommendation, ensuring the standardization and homogenization of radiotherapy and achieve the individualized intensity-modulated radiotherapy for nasopharyngeal carcinoma patients.

Objective:To study the distribution of pathogenic microorganisms in the ocular fluid of patients with acquired immunodeficiency syndrome (AIDS) and infectious uveitis.Methods:It was a retrospective case analysis. From June 2018 to December 2019, 31 AIDS patients with infectious uveitis who were hospitalized or outpatient at Shanghai Public Health Clinical Center were included in the study. Among them, there were 30 males and 1 female; the average age was 38.51±11.17 years. There were 20 cases of panuveitis, 10 cases of posterior uveitis, and 1 case of infectious endophthalmitis. Serum CD4 +T lymphocyte count (CD4 +TC) were 0 - 239/μl during the same period. The second-generation gene sequencing technology was used to detect the collected intraocular fluid. Among 31 specimens, aqueous humor and vitreous humor were 27 and 4 respectively. Results:Among 31 specimens, 18 samples (58.1%, 18/31) of cytomegalovirus (CMV) were detected; varicella-zoster virus (VZV) were detected in 5 samples (16.1%, 5/31); Epstein-Barr virus were detected in 9 samples (29.0%, 9/31); human beta herpes virus type 6 (HHV6) were detected in 3 samples (9.7%, 3/31), human papillary molluscum virus (HPV), human polyoma virus, type G hepatitis virus were separately detected in 1 sample (3.2%, 1/31), all coexisting with other microorganisms. Parvovirus were detedcted in 8 samples (25.8%, 8/31); treponema pallidum were detedcted in 5 samples (16.1%, 5/31); toxoplasma gondii and Harmon coccidia were detedcted in 1 sample (3.2%, 1/31); synitelium Polycarpum were detedcted in 1 sample (3.2%, 1/31); mycobacterium tuberculosis complex, fungi, and microbacteria coexist were detedcted in 1 sample (3.2%, 1/31). Among the 18 CMV specimens, the number of gene sequences was more than 1059 (50.0%), and 104-1055 (27.7%). Among the 5 specimens of VZV, the number of gene sequences was>1044 (80.0%). In one specimen, the mycobacterium tuberculosis complex, fungi, and microbacteria coexist, and the number of gene sequences were all <100. The number of gene sequences of HHV6, HPV, human polyoma virus, type G virus, and parvovirus in all specimens was small. Among 31 specimens, 15 (48.4%) of pathogenic microorganisms were detected at least 2 species.Conclusions:CMV and VZV are the main pathogenic microorganisms of infective uveitis in patients with serum CD4 +TC <100/μl; treponema pallidum, toxoplasma gondii or other protozoa, mycobacterium tuberculosis, and fungi cause more infectious uveitis which are common in AIDS patients with serum CD4 +TC >100/μl. The coexistence of two or more microorganisms can be detected in the intraocular fluid of AIDS patients with infectious uveitis.