According to the amino acid sequence of monellin, a single chain 294bp monellin gene was synthesized and inserted into vector pET-22b to yield the recombinant secretion plasmid pETMO. The single-chain monellin gene was designed based on the biased codons of E. coli so that its expression would be then optimized. Under the expressing conditions, monellin was produced accounting for 44.8% of total soluble proteins. The E. coli-expressed single-chain monellin is 3000 times sweeter than sucrose. The thermal-stability and acid-resistance of the protein are higher than the natural monellin.
Escherichia coli ; genetics ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo present an established human chordoma cell line for chordoma research.

METHODSThe specimens pathologically identified as chordoma were cultured, using primary tissue culture in vitro. The surviving cells were analyzed by morphology, histochemical stain, cell cycling analysis, karyotype analysis, electron microscopic observation, heterotransplantation and study of invasive capacity in vitro.

RESULTSThe newly established cell line CM-319 has been maintained in continual cultures for over 100 generations in two years. Its morphological observation, histochemical staining properties, electron microscopic observation and heterotransplantation showed the common characteristics of chordoma. The doubling time of cells was about 33 hours. Cell cycle analysis showed: G(1) 55.6%, G(2) 21.9% and S 22.5%, G(2)/G(1) = 1.90. Chromosome analysis showed a hypotriploid feature and the success rate of heterotransplantation was 100%. It is capable of invasion in vitro.

CONCLUSIONCM-319, as a cell line derived from human chordoma cells, may serve for further studies of chordoma.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Chordoma ; genetics ; pathology ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Invasiveness

OBJECTIVETo investigate the status of c-kit and PDGFRA mutations of GIST in a the large sample of Chinese patients.

METHODOne hundred and sixty-five cases were evaluated for the presence of c-kit and PDGFRA mutations. Exon 9, 11, 13, 17 of c-kit and exon 12, 18 of PDGFRA were analyzed by PCR amplification and direct sequencing.

RESULTSImmunohistochemical demonstrations of KIT (CD117) were seen in 94% of the cases (155/165). Overall, c-kit mutations were identified in 76.1% (118/155) of CD117 positive cases: 67.1% (104/155) involving exon 11, 7.1% (11/155) involving exon 9, 1.3% (2/155) involving exon 13 and 0.6% (1/155) involving exon 17. The c-kit exon 11 mutations were mostly heterogeneous and clustered in the classic "hot spot" at the 5' end of the exon, including in-frame deletion and point mutation. The second "hot spots" were internal tandem duplications (ITD) at the 3' end of the exon, which were associated with female patient, older age, stomach location and low mitotic counts. The exon 9 mutations correlated with a distinct subset of GISTs involving the small bowel of young male patients. A new point mutation of L641P was identified in exon 13. PDGFRA mutations were present in 50% (5/10) of CD117-negative GISTs, all involving exon 18 with the majority of mutations being D842V. One novel in-frame deletion of IMHD mutation at codon 843 - 846 with S847T was identified. GISTs with PDGFRA mutations were often larger tumors arising from the omentum/mesentery of young male patients with high risk of aggressive behavior.

CONCLUSIONSThe vast majority of GISTs in this study harbored c-kit and PDGFRA mutations, there were non-random relations between the gene mutation patterns and the locations of GISTs. It appears that Chinese GIST patients have some unique mutation patterns. It is necessary to evaluate the gene mutations status of GISTs to guide target therapy.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Amino Acid Sequence ; Base Sequence ; Child ; DNA Mutational Analysis ; DNA, Neoplasm ; chemistry ; genetics ; Exons ; genetics ; Female ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; Sequence Homology, Amino Acid

Objective To investigate the effect and its mechanism of ziyuglycoside Ⅱ on prolifer-ation,migration,invasion and apoptosis of colon cancer cells HT-29.Methods The effect of ziyugly-coside n on cell proliferation of colon cancer cells HT-29 was determined by CCK-8 method;the effect of ziyuglycoside Ⅱ on cell migrative capacity of colon cancer cells HT-29 was determined by scratch assay;the effect of ziyuglycoside n on cell invasive capacity of colon cancer cells HT-29 was determined by transwell assay;the effects of ziyuglycoside n on cell apoptosis of colon cancer cells HT-29 was determined by flow cytometry;the effects of ziyuglycoside Ⅱ on mRNA and protein expres-sion of protein kinase B(AKT)/phosphatidylinositol-3-kinase(PI3K)signal pathway were determined by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and western-blot,respectively.Results Ziyuglycoside n(0,1,5,10,20,40,60 and 80 μmol/mL)inhibited pro-liferation of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside n(5,10 and 20 μmol/mL)inhibited migration of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglyco-side Ⅱ(5,10 and 20 μmol/mL)inhibited invasion of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)promoted apoptosis of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)increased mRNA expression of AKT and PI3K,decreased mRNA expression of Caspase-3 and Caspase-9.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)increased protein expression of phosphorylated protein kinase B(p-AKT)and phosphorylphosphatidylinositol-3-kinase(p-PI3K),increased the expression of Cleaved-Caspase-3 and Cleaved-Caspase-9 protein.Conclusion Ziyuglycoside Ⅱ can inhibit proliferation,migration and invasion of colon cancer cells HT-29,and promote the apoptosis of colon cancer cells HT-29.Its mechanism may be related to regulating the signal pathway of AKT/PI3K via promoting phospho-rylation of AKT and PI3K protein,activation of Caspase-3 and Caspase-9 protein.

Objective:To investigate whether stress hyperglycemia (SH) is an independent risk factor for the occurrence and mortality of sepsis-associated encephalopathy (SAE).Methods:From August 2016 to October 2021, sepsis patients admitted to the ICU of Guangdong Provincial People's Hospital were selected as the study subjects. According to whether they developed to SH (RBG>11.1 mmol/L) within 7 days of enrollment, the pat ients were divided into the SH group and the non-SH group for analysis. Logistic regression was used to analyze whether SH was an independent risk factor for SAE occurrence, and ROC curve was used to analyze the predictive value of SH to SAE. Kaplan-Meier curve was used to compare the 90-day survival of SAE patients with or without SH. Cox regression analysis was used to analyze the risk factors of 28-day and 90-day death in SAE patients.Results:A total of 183 sepsis patients were included, including 62 patients in the SH group and 121 in the non-SH group. Logistic regression analysis demonstrated that SH was an independent risk factor for SAE ( OR=4.452, 95% CI: 2.021-9.808, P <0.001). ROC curve demonstrated that SH could accurately predict SAE (AUC=0.831; Sensitivity=78.4%; Specificity=76.8%; and Yoden index=0.553). Kaplan-Meier curve demonstrated that the 90-day survival of SAE patients with SH significantly declined (log-rank test: P<0.01). Cox regression analysis suggested that SH was a risk factor for death at day 28 and day 90 in SAE patients (28 d, HR=2.272, 95% CI: 1.212-4.260, P=0.010; 90 d, HR=2.456, 95% CI: 1.400-4.306, P<0.01). Conclusions:SH is an independent risk factor for SAE and can predict SAE occurrence. SH significantly reduces 90-day survival and increase mortality at 28 and 90 days in SAE patients.

Objective To investigate the effect and its mechanism of ziyuglycoside Ⅱ on prolifer-ation,migration,invasion and apoptosis of colon cancer cells HT-29.Methods The effect of ziyugly-coside n on cell proliferation of colon cancer cells HT-29 was determined by CCK-8 method;the effect of ziyuglycoside Ⅱ on cell migrative capacity of colon cancer cells HT-29 was determined by scratch assay;the effect of ziyuglycoside n on cell invasive capacity of colon cancer cells HT-29 was determined by transwell assay;the effects of ziyuglycoside n on cell apoptosis of colon cancer cells HT-29 was determined by flow cytometry;the effects of ziyuglycoside Ⅱ on mRNA and protein expres-sion of protein kinase B(AKT)/phosphatidylinositol-3-kinase(PI3K)signal pathway were determined by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and western-blot,respectively.Results Ziyuglycoside n(0,1,5,10,20,40,60 and 80 μmol/mL)inhibited pro-liferation of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside n(5,10 and 20 μmol/mL)inhibited migration of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglyco-side Ⅱ(5,10 and 20 μmol/mL)inhibited invasion of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)promoted apoptosis of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)increased mRNA expression of AKT and PI3K,decreased mRNA expression of Caspase-3 and Caspase-9.Ziyuglycoside Ⅱ(5,10 and 20 μmol/mL)increased protein expression of phosphorylated protein kinase B(p-AKT)and phosphorylphosphatidylinositol-3-kinase(p-PI3K),increased the expression of Cleaved-Caspase-3 and Cleaved-Caspase-9 protein.Conclusion Ziyuglycoside Ⅱ can inhibit proliferation,migration and invasion of colon cancer cells HT-29,and promote the apoptosis of colon cancer cells HT-29.Its mechanism may be related to regulating the signal pathway of AKT/PI3K via promoting phospho-rylation of AKT and PI3K protein,activation of Caspase-3 and Caspase-9 protein.

OBJECTIVE: To observe the effect of staged acupuncture on serum irisin level, neurological deficit, balance ability and spasticity in patients with ischemic stroke. METHODS: Sixty patients with ischemic stroke were randomly divided into a staged acupuncture group and a routine acupuncture group, 30 cases in each group; another 30 healthy subjects were selected as a normal group. The patients with ischemic stroke were treated with aspirin (100 mg each time, once a day, changing to 50 mg for prophylactic dose after 4 weeks). The patients in the staged acupuncture group were treated with staged acupuncture (acupoints were selected according to the soft paralysis period, spasticity period and recovery period, sequelae period) and rehabilitation treatment, while the patients in the routine acupuncture group were treated with acupuncture of soft paralysis-period as the staged acupuncture group and rehabilitation treatment. All the treatment was given once a day, 5 times a week, 2 weeks as a course of treatment, and 4 consecutive courses of treatment were provided. Before treatment and at 2 weeks, 4 weeks, 6 weeks and 8 weeks into treatment, the serum irisin level was measured, and the scores of National Institutes of Health stroke scale (NIHSS), Fugl-Meyer assessment scale-balance (FM-B) and comprehensive spasticity scale (CSS) were compared, and the correlation between the serum irisin level and NIHSS and FM-B scores in the two groups was analyzed. RESULTS: Before treatment, the serum irisin levels in the two groups were lower than those in the normal group (P<0.01). Compared before treatment, the serum irisin levels and FM-B scores were increased (P<0.01), and the NIHSS scores were decreased at 2, 4, 6 and 8 weeks into treatment in the two groups (P<0.01). At 4, 6 and 8 weeks into treatment, in the staged acupuncture group, the serum irisin levels and FM-B scores were higher than those in the routine acupuncture group (P<0.01, P<0.05), and the NIHSS scores were lower than those in the routine acupuncture group (P<0.01). After treatment, the CSS scores in the two groups were increased first and then decreased. Compared before treatment, the CSS scores were increased at 2, 4, 6 and 8 weeks into treatment in the two groups (P<0.01). At 4, 6 and 8 weeks into treatment, the CSS scores in the staged acupuncture group were lower than those in the routine acupuncture group (P<0.01). The serum irisin level was negatively correlated with NIHSS score (r =-0.772, P =0.000), and positively correlated with FM-B score (r =0.675, P =0.000). CONCLUSION The severity of neurological deficit and balance ability are related to serum irisin level in patients with ischemic stroke. The staged acupuncture could increase the serum irisin level, improve the neurological function, balance ability and spasticity in patients with ischemic stroke.
Acupuncture Therapy ; Fibronectins ; Humans ; Ischemic Stroke ; Muscle Spasticity ; Neurological Rehabilitation ; Paralysis/complications* ; Stroke/therapy* ; Stroke Rehabilitation ; Treatment Outcome

OBJECTIVE To explore the efficacy of Sour Jujube Seed Decoction combined with Huanglian Wendan Decoction on adult chronic insomnia and its effect on hypnotic withdrawal.METHODS 187 patients with chronic insomnia were included for anal-ysis,including 102 in the traditional Chinese medicine(TCM)group and 85 in the western medicine group.The TCM group was trea-ted with Sour Jujube Seed Decoction combined with Huanglian Wendan Decoction,while the western medicine group was treated with benzodiazepine under the consideration of doctor.The intervention period was 1 month,with assessments using the Pittsburgh Sleep Quality Index(PSQI)conducted before and after the intervention.Follow-up evaluations were performed at 3 months and 6 months re-spectively after the intervention.RESULTS There was no significant difference between the two groups at baseline.After the inter-vention,the PSQI scores of patients in both groups were significantly improved(P<0.01).Among them,the TCM group was better than the western medicine group in the improvement of sleep quality and sleeping pills,total PSQI score reduction(P<0.01).The re-sults of linear regression analysis showed that after controlling for confounding factors,the regression coefficients of the TCM group in two different models were1.821 and 1.922 respectively,and the former was statistically significant(P<0.05).By screening patients who took hypnotics at baseline in the TCM group and comparing them with those in the western medicine group,the influencing factors of hypnotic withdrawal were analyzed.During the 3-month follow-up,25 out of 39 patients in the TCM group and 17 out of 80 patients in the western medicine group had hypnotic withdrawal(χ2= 19.25,P<0.001);during the 6-month follow-up,23 of the 39 patients in the TCM group and 18 of the 79 patients in the western medicine group had hypnotic withdrawal(χ2= 13.53,P<0.001),the with-drawal rate of patients in the TCM group was significantly higher than that in the western medicine group.Further regression analysis showed that after adjusting for confounding factors,the results showed that the western medicine group had a significantly higher rate of not withdrawal than the TCM group at 3 months(OR=5.50,95%CI:2.30～13.72)and 6 months(OR=6.43,95%CI:2.54～17.77),and the results were statistically different(P<0.05).CONCLUSION Sour Jujube Seed Decoction combined with Huangli-an Wendan Decoction is effective in treating adult chronic insomnia and assisting in hypnotic withdrawal.

OBJECTIVEIn mid-July 2005, five patients presented with septic shock to a hospital in Ziyang city in Sichuan, China, to identify the etiology of the unknown reason disease, an epidemiological, clinical, and laboratory study were conducted.

METHODSAn enhanced surveillance program were established in Sichuan, the following activities were introduced: active case finding in Sichuan of (a) laboratory diagnosed Streptococcus suis infection and (b) clinically diagnosed probable cases with exposure history; supplemented by (c) monitoring reports on meningococcal meningitis. Streptococcus suis serotype 2 infection was confirmed by culture and biochemical reactions, followed by sequencing for specific genes for serotype and virulence factors.

RESULTSFrom June 10 to August 21, 2005, 68 laboratory confirmed cases of human Streptococcus suis infections were reported. All were villagers who gave a history of direct exposure to deceased or sick pigs in their backyards where slaughtering was performed. Twenty six (38%) presented with toxic shock syndrome of which 15 (58%) died. Other presentations were septicaemia or meningitis. All isolates were tested positive for genes for tuf, species-specific 16S rRNA, cps2J, mrp, ef and sly. There were 136 clinically diagnosed probable cases with similar exposure history but incomplete laboratory investigations.

CONCLUSIONAn outbreak of human Streptococcus suis serotype 2 infections occurred in villagers after direct exposure to deceased or sick pigs in Sichuan. Prohibition of slaughtering in backyards brought the outbreak to a halt. A virulent strain of the bacteria is speculated to be in circulation, and is responsible for the unusual presentation of toxic shock syndrome with high case fatality.

Animals ; Bacteremia ; epidemiology ; microbiology ; China ; epidemiology ; Disease Outbreaks ; Humans ; Meningitis, Bacterial ; epidemiology ; microbiology ; Shock, Septic ; epidemiology ; microbiology ; Streptococcal Infections ; epidemiology ; microbiology ; veterinary ; Streptococcus suis ; isolation & purification ; Swine ; Swine Diseases ; microbiology

Artificial Intelligence ; Humans ; Pulmonary Disease, Chronic Obstructive/diagnosis*