Objective To study the role of protective antigen(PA)and N-terminal segment of lethal factor (LFn)in the entrance of EGFP(enhanced green fluorescent protein)into HeLa cells. Methods The DNA fragments encoding LFn and EGFP were amplified,respectively,and cloned into the plasmid pET-21 a(+)one after another to construct a recombinant plasmid pET-LFn-EGFP. The plasmid was txansformed into BL21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubation with LFn-EGFP in the presence of PA or not, the HeLa cells were analyzed by flow cytometry or laser confocal microscopy. Results The fusion protein LFn-EGFP was purified by over 90% homogeneity and retained the ability of LF to bind with PA when incubated with J774A.1 macrophage cells,and could get into HeLa cells. Conclusion The LFn-EGFP could enter the HeLa cells in a PA independent pathway. But PA could help more LFn-EGFP molecules enter into HeLa cells.

OBJECTIVETo evaluate the clinical effectiveness and safety of intubation-surfactant-extubation (INSURE) method in the treatment of neonatal respiratory distress syndrome (NRDS), and to investigate its possible mechanisms.

METHODSSixty-four premature infants, who were admitted for NRDS and treated with pulmonary surfactant from March 2010 to March 2012, were enrolled in the study. They were randomly divided into INSURE (n=32) and conventional mechanical ventilation (CMV) groups (n=32). The two groups were compared in terms of respiratory function, ventilation time, duration of oxygen therapy, complications, and prognosis, as well as expression of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and serum ferritin (SF).

RESULTSOxygenation index in the INSURE group was significantly higher than in the CMV group at 48 hours after treatment (P<0.05). Compared with the CMV group, the INSURE group showed significantly lower incidence of ventilator-associated pneumonia (VAP) and significantly shorter duration of oxygen therapy (P<0.05 for all comparisons). There were no significant differences in ventilation time and the incidence of pneumothorax, intracranial hemorrhage, necrotizing enteroolitis, bronchopulmonary dysplasia, and pneumorrhagia between the two groups (P>0.05). The levels of TNF-α and SF were significantly lower in the INSURE group than in the CMV group at 6, 24, 48, and 72 hours after treatment (P<0.05), while the level of IL-10 was significantly higher in the INSURE group than in the CMV group (P<0.05).

CONCLUSIONSINSURE method can improve the oxygenation function of the lung, decrease the incidence of VAP and shorten the duration of oxygen therapy in neonates with NRDS, which is probably due to the fact that this method can reduce the production of TNF-α and SF and inhibit the decrease of IL-10.

Carbon Dioxide ; blood ; Female ; Ferritins ; blood ; Humans ; Infant, Newborn ; Interleukin-10 ; blood ; Male ; Respiration, Artificial ; Respiratory Distress Syndrome, Newborn ; blood ; therapy ; Tumor Necrosis Factor-alpha ; blood

Objective To investigate the distribution characteristics and risk factors of intracranial atherosclerotic stenosis ischemic stroke. Methods We retrospectively collected 342 consecutive patients with first-ever ischemic stroke. Clinical data was collected including demographics, the presence of risk factors,MRI with MRA and other routine admis?sion laboratory tests. Results Intracranial atherosclerotic stenosis (ICAS) was located most frequently in MCA (47.0%), Extracranial internal carotid artery was the most common affected artery (65.0%) among extracranial atherosclerotic steno? sis (ECAS). MetS (OR=1.586,95%CI:1.232~2.268), ApoB/ApoA1 ratio (OR=1.926,95%CI:1.051~4.288), were as?sociated with ICAS (vs ECAS), whereas hypertension (OR=3.603,95%CI:1.675~12.485), MetS (OR=2.268,95%CI:1.274~6.103), HbA1c (OR=2.015,95%CI:1.182~5.613) and ApoB/ ApoA I ratio (OR=1.948,95%CI:1.157~4.285) were related to ICAS (vs NCAS). Hypertension (OR=2.437,95%CI:1.492~3.505,P=0.005), Hcy (OR=2.437,95%CI:1.492~3.505,P=0.005) and HbA1c (OR=1.769,95%CI:1.034~3.121, P=0.005) were the independent risk factors re?lated to posterior circulation strokes (vs anterior circulation strokes ) in ICAS strokes. Conclusions The occurrence of ICAS may be more frequent than that of ECAS in ischemic stroke. Posterior circulation ICAS strokes seems to be close?ly associated with metabolic derangement.

Objective To investigate the expression of intestinal epithelial stem cell markers Msil and Hesl in mouse acute radiation enteritis (RE) model. Methods Forty female BALB/c mice were undergone one-time whole abdominal irradiation by 300 cGy/min β-ray. Thirty mice were selected before irradiation (Day 0) and every six mice were euthanized at day 3,5,7 and 14 after irradiation. The small intestinal were detached and the expression of Msil and Hesl were detected in middle part of small intestinal by quantitative RT-PCR, Western blots, and immunohistochemistry. Subsequently,the difference of Msil and Hesl expression was compared. Results The mouse acute RE model was successfully established. At the 5th day after radiation, the results of quantitative RT-PCR indicated that the relative ΔCt value of Msil and Hesl expression were 15. 17 ± 0. 47 and 15. 83 ± 0. 24respectively, the results of Western blot showed that the integrated intensity were 0. 443±0. 055 and 0. 301 + 0. 047 for Msil and Hesl. The expressions were significantly higher than other time points at RNA and protein levels (P<0. 05). The results of immunohistochemistry revealed that Msil and Hesl were highly expressed in intestinal crypts at 5th day and 7th day. Conclusions The expression of Msil and Hesl, upregulated before wound repair in RE model, indicates that intestinal epithelial repair is related to intestinal stem cell proliferation.

Objective To investigate the role of Notch signaling pathway in cerebral ischemic tolenmce induced by clectroacupuncture (EA) preconditioning.Methods Fifty-two adult male SD rats weighing 280-320 g were randomly divided into 2 groups(n=each):control group(group C)and electroacupuncture preconditionig group(group EA).Group C received no treatment.Group EA received EA at the Baihui acupoint (GV20) for 30 min a day for 5 days.Twenty-four hours after the last preconditionig,focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min,followed by 72 h of reperfusion.Notch intracellular domain(NICD)expression was determined by Western blot and expression of Notch1,Notch4,Jag1,and Hes1 mRNA by real-time PCR immediately before iachemia and 24 and 72 h of reperfusion.The neurological deficit was scored at 72 h of reperfusion.The infarct volumes were then determined after evaluation of the neurological deficit score .Results There was no significant difference in Hesl mRNA and NICD expression immediately before ischemia between group EA and C(P>O.05).NICD expression was up-regulated at 24 and 72 h of reperfusion in both groups, and Hesl mRNA expression at 72 h of reperfusion in group C and at 24 h of reperfusion in group EA was up-regulated compared with those immediately before ischemia (P < 0.05). Hes1 mRNA and NICD expression was up-regulated in group C, while down-regulated in group EA at 72 h of reperfusion compared with those at 24 h of reperfusion ( P < 0.05 ). Compared with group C, the expression of Notchl,Notch4 and Jag1 mRNA was up-regulated immediately before ischemia, and Hes1 mRNA and NICD expression was up-regulated at 24 h of reperfusion while down-regulated at 72 h of reperfusion in group EA( P < 0.05). EA preconditioning significantly reduced infarct volumes and increased neurological deficit scores at 72 h of reperfusion (P < 0.05 ). Conclusion Notch signaling pathway may participate in cerebral ischemic tolerance induced by EA preconditioning.

Objective: To make etiological diagnosis and evaluate the protective effects of post-exposure prophylaxis(PEP) in an event of one dog injured seven persons. Methods: Direct immunofluorescence assay (DFA) and nested polymerase chain reaction (PCR) were employed to detect nucleoprotein and nucleoprotein(N) gene of rabies virus in the brain tissues of the dog, the positive samples were sequenced for the full length of N gene of rabies virus, then the homology of the N gene of rabies virus was analyzed after the phylogenetic tree was constructed. Rapid fluorescent focus inhibition test (RFFIT) was applied to detect the rabies virus neutralizing antibodies(RVNA) on day 0, 14 and 40 after PEP. Results: The cerebral, cerebellar and hippocampal tissues were positive by DFA and nested PCR. The phylogenetic tree indicated the rabies virus belonged to the rabies virus genotype I. The homology of the nucleotide and amino acid of the rabies virus N gene were over 86% with the vaccine strains. The titer of the RVNA increased significantly from the day 0 to day 14 after PEP, the lowest was 5.78 IU/ml and the highest was 26.15 IU/ml. On the day 40, the highest RVNA titer was 51.96 IU/ml. No rabies cases occurred in a one year follow-up visit. Conclusions Normative PEP can effectively prevent the occurrence of rabies cases.

OBJECTIVES: There is less clinical data on multiple myeloma (MM) in China, and the aim of this study was to collect and analyze the clinical data of newly diagnosed multiple myeloma (NDMM) patients in Hunan Province during 1 year, to understand the real clinical features and treatment outcome for Hunan Province patients with MM, and to strengthen the understanding of the standardized diagnosis process and treatment plan of MM. METHODS: The clinical data of 529 patients with NDMM in 12 large-scale general hospitals in Hunan Province from January 1 to December 31, 2019 were collected and analyzed, including baseline data, treatment regimens, duration of treatment, and adverse reactions. The clinical characteristics, treatment, and safety of patients were analyzed by SPSS 21.0. RESULTS: Among the 529 NDMM patients, the age was 33-90 (median 64) years and the male-female ratio was 1.38꞉1. The clinical features ranged from high to low were as follows: Bone pain (77.7%), anemia (66.8%), renal insufficiency (40.6%), hypercalcemia (15.1%). Typing: IgG 46.5%, IgA 24.6%, IgD 2.6%, IgM 0.8%, light chain 15.7%, double clone 3.0%, no secretion 0.6%, absence 6.2%. Staging: Durie-Salmon stage I, II, and III were 4.5%, 10.6%, 77.3%, respectively, and 40 cases (7.6%) missed this data. International Staging System (ISS) stage I, II, and III were 10.4%, 24.4%, and 47.6%, respectively, and 93 cases (17.6%) were missing. Revised International Staging System (R-ISS) stage I, II, and III were 5.5%, 27.0%, 23.1%, respectively, and 235 cases (44.4%) missed this data. Among the 98 NDMM patients in the Third Xiangya Hospital, Central South University, Durie-Salmon (DS) stage missing 2.0%, ISS stage missing 12.3%, and R-ISS stage missing 12.3%.Treatment: Among the 529 patients,475 received treatment, the rate of treatment was 89.8%; 67.4% of the patients were able to complete four courses of chemotherapy at induction phase, 90.3% of the patients received proteasome inhibitor based combination chemotherapy regimen more than once, 67.2% received immunomodulator based regimen more than once, and 59.8% of the patients received proteasome inhibitor and immunomodulator based combination chemotherapy regimen more than once. Curative: Overall response rate (ORR) and high quality response rate (HQR) of the 4-course group were better than those of the 2-course group (ORR: 85% vs 65%, P=0.006; HQR: 68.3% vs 24.0%, P<0.001). The HQR of the standard chemotherapy group was better than that of the non-standard chemotherapy group (65.1% vs 48.2%, P=0.035). Adverse reactions during treatment included hematologic toxicity (17.5%), peripheral neuropathy (24.8%), gastrointestinal adverse events (23.8%), pulmonary infection (25.9%), herpes zoster (4.6%), and venous thrombotic events (1.7%). CONCLUSIONS In 2019, the missed diagnosis rate of MM patients was high, the medium age of diagnosis was older, and the accuracy of patient diagnosis was not high. There is a great difference among medical centers, especially in the stage and risk stratified, nearly half of NDMM patients are not diagnosed with R-ISS stage; the lack of cytogenetic data needs to be supplemented by follow-up studies. A high proportion of patients with NDMM present with bone pain and anemia.Patients received treatment have higher use of chemotherapy regimens containing proteasome inhibitors and/or immunomodulators, but there is a significant gap among different medical centers, and standardized treatment needs to be strengthened. The safety during chemotherapy is controllable.
Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Female ; Humans ; Immunologic Factors/therapeutic use* ; Male ; Middle Aged ; Multiple Myeloma/therapy* ; Neoplasm Staging ; Pain ; Prognosis ; Proteasome Inhibitors/therapeutic use*