Not only calmodulin (CAM) with Ca2+ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca2+bound) and Ca2+-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca2+-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM21, mCoM212,mCaM2123, mCaM2124 and mCaM21234 were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca2+-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca2+ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. 45Ca2+ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca2+ in the mCaM2 proteins. The mCaM21234 mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca2+. Using yeast two-hybrid technique with mCaM21234 as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca2+, CaM and Ca2+-independent CaMBPs in plant biological processes.

OBJECTIVE:To investigate the effects of uterine arterial embolization combined with Methotrexate and Lactate ethacri-dine on related indexes of mid-pregnant patients with placenta previa abortion. METHODS:The data of 100 mid-pregnant patients with placenta previa who required inducing labor was retrospectively analyzed and divided into observation group(50 cases)and control group(50 cases)by different treatment. Control group received uterine arterial embolization before inducing labor,then received Lac-tate ethacridine injection 100 mg,amniotic injection+Mifepristone tablet 75 mg,and aborted after 2 d. Observation group received uterine arterial embolization after uterine arterial perfusionon both sides,then received Methotrexate for injection 50 mg/side after uter-ine arterial embolization catheter entering uterine artery,Lactate ethacridine injection(the same usage and dosage as control group)+Mifepristone tablet(the same usage and dosage as control group)was given after 12 h. Total parturition,intrapartum hemorrhage,hos-pitalization time,menstruation recovery time,cesarean section,hysterectomy,retained placenta and clearing palace in 2 groups were observed,and postoperative complications were recorded. RESULTS:Intrapartum hemorrhage,hospitalization time,cesarean section rate and hysterectomy rate in observation group were significantly lower than control group,with statistical significance(P<0.05), while there were no significant differences in total parturition,menstruation recovery time,retained placenta rate and clearing palace rate in 2 groups(P>0.05). There was no significant difference in the incidence of postoperative complications in 2 groups(P>0.05). CONCLUSIONS:Uterine arterial embolization combined with Methotrexate and Lactate ethacridine can effectively reduce the hemor-rhage of mid-pregnant patients with placenta previa abortion,shorten hospitalization time,and decrease cesarean section and hysterec-tomy rate.

AIM:To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS:Sequences of TLR9 siRNAs were designed.A549 and H520 cells were transfected with TLR9 siR-NA by lipofectamine.The expression of TLR9 was detected by Western blot .The cell activity was measured by CCK-8 as-say.The experiments were divided into blank control group , control siRNA group and TLR9 siRNA interference group.The cell cycle distribution and cell apoptosis were analyzed by flow cytometry .The expression of P38 and Bax was determined by Western blot .The cells in each group were exposed to CpG ODN 7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity , G2/M phase arrest and apoptosis , but in-creased the protein expression of P 38 and Bax ( P<0.01) .In addition, there was no significant changes of the above inde-xes in CpG ODN7909 treated-TLR9 siRNA group was observed .DOC alone significantly inhibited the cell activity , higher the G2/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn’t affect the expression of P38 in all 3 groups.Compared with the cells treated with DOC alone , the cells treated with CpG ODN7909 combined with DOC exhibi-ted lower cell activity, higher G2/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no sig-nificant change of P38 expression.In addition, there was no significant change of the above indexes in CpG ODN 7909 com-bined with DOC treated-TLR9 siRNA group was observed .CONCLUSION:CpG ODN7909 may enhance the chemothera-peutic sensitivity of DOC in human lung cancer cells by combining with TLR 9.The mechanism might be related to enhan-cing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G 2/M phase of the cells .

Genome of Arabidopsis has a kind of genes encoding proteins with ARM repeat domains and some of these proteins are known to play important roles in plant development and responses to hormone. An Arabidopsis mutant lfr with a distinct phenotype was got in leaf and flower development. The gene is predicted to encode a protein with ARM repeat domains. In order to study its function and molecular mechanism, recombination expression plasmid pGEX-2TGST∶LFR was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The expression products were detected by 12% SDS-PAGE. The GST∶LFR fusion protein was existed in soluble form with a relative molecular mass 77 ku, which is fit with the molecular mass supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 ku, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.

[ ABSTRACT] AIM:To explore the mechanisms of fluctuant high blood glucose-induced apoptosis of hepatocytes. METHODS:SD rats were randomly divided into normal control group ( N) , stable high blood glucose group ( S) , fluctu-ant high blood glucose group ( F) and insulin group ( I) .Diabetic rats were induced by intraperitoneal injection of strepto-zotocin (65 mg/kg) , and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day.The blood glucose fluctuation patterns of the animals in F group with-in 12 weeks were similar every day and no significant difference of the HbA1c concentration was observed compared with S group, indicating that the fluctuant hyperglycemia was successfully established in F group.The activity of superoxide dis-mutase ( SOD) and glutathione peroxidase ( GSH-Px) , and the content of malondialdehyde ( MDA) and nitric oxide ( NO) in the homogenate of the liver tissues were detected by colorimetry.The mRNA and protein levels of JNK, p-JNK, Bax and Bcl-2 were examined by RT-PCR and Western blot.RESULTS:After 12 weeks, the increases in the intakes of food and water, the urine output, and the abnormal liver function were observed in S group, I group and F group.Compared with N group, the MDA level was increased, the content of NO and the activity of SOD and GSH-Px were decreased, and up-regu-lation of JNK mRNA and p-JNK and Bax proteins, and down-regulation of Bcl-2 were also found in S group, I group and F group.The above effects were more obviously showed in F group.CONCLUSION:Oxidative stress activates JNK-MAPK signaling pathway, which is involved in fluctuant high glucose-induced apoptosis of hepatocytes.

Objective To explore the correlation between environmental selenium(Se) levels and cognitive ability among rural elderly population, and the effect of Se on cognitive skill of the elderly. Methods Two study sites(Zichuan district and Gaomi city of Shandong) with different environmental Se levels were selected according to rural elderly people ≥ 65 years were extracted by stratified random sampling method in each site. A retrospective survey was carried out using dietary intake questionnaire for the elderly for the past 1 year, and their daily total Se intake was calculated. Questionnaire was also used to obtain cognitive skill information in the elderly people. The Community Screening Instrument for Dementia, the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List Learning Test, the Indiana University (IU) Story Recall Test, the Animal Fluency Test, and the IU Token Test were applied to assess the cognitive ability. Se level in these samples was analyzed. Correlation between the Se levels of environment and those of human body were analyzed statistically. Results There were 0.163), (0.405 ± 0.086)]mg/kg and nail Se[(147.44 ± 17.42), (117.38 ± 22.48)μg/L]between the two groups (U = 31.59, 25.00, 23.67, all P ＜ 0.01 ). There were positive correlation among the nail Se, environmental Se and Se in all subjects(r = 0.51,0.46, 0.60, all P ＜ 0.01 ). The differences of the CSID total score, the CERAD Word List Learning Test, the CERAD Word List Recall Test and the IU Story Recall Test between the two sites were statistically significant(F = 2.56, 9.18, 7.48, 4.42, all P ＜ 0.05), excluding the Indiana University Story Recall Test.After eliminating possible confounding factors, the Se levels and the CSID total score, the IU Story Recall Test, the Animal Fluency Test and the IU Story Token Test had a significantly positive correlation (r = 0.076, 0.138, 0.042,0.107, P ＜ 0.05 or ＜ 0.01 ), excluding the CERAD Word List Learning Test. Conclusions This study supports the hypothesis that a life long low Se level is associated with lower cognitive ability. The cognitive abilities in the elderly population lived in areas with high environmental selenium levels are significantly higher than that of the elderly lived in areas with low environmental selenium levels.

Objective To assess the prognosis and effect on renal function of pediatric urolithiasis caused by melamine-contaminated milk powder (PUMMP) in a long-term follow-up.Methods One hundred and two of 8335 children (≤ 6-year-old) with history of consuming melamine-contaminated milk powder screened in our hospital were followed up for eighteen months after diagnosis. Urinary system ultrasonography, urinalysis, urinary microprotein profiles [microalbumin (ALBU), immunoglobulin G (IgG), and N-acetyl-β-D-glucosidase (NAG)], urinary melamine and cyanuric acid were examined in the first visit and at the end of follow-up. Results Follow-up was completed in 91 children and the stone was excreted in 82 children (90.1%).Stones less than 5 mm in diameter were most vulnerable to discharge, and stones larger than 10 mm could not be expelled without interventions. At the end of follow-up, no melamine or cyanuric acid was found in the urine samples of 74 patients. Urinalysis showed that incidences of proteinuria, microscopic hematuria and leukocyturia were 0%, 5.1% and 2.0%, which were significant different from those in the first visit (Pproteinutria=0.123, Phemnatuna=0.038 and Pleukocyhuris=0.005).Urinary microprotein profiles revealed that some children whose urinalysis was normal still presented glomerular and renal tubular injury and the abnormal rates were 8.8% and 12.1%respectively. The glomerular injury was mainly related to persistent stone, male and younger.Conclusions 90.1% of children with PUMMP passes urinary stones at the end of follow-up.Stone size is the major risk factor of discharge. No melamine or cyanuric acid is found in the urine of children. After eighteen months, glomerular and renal tubular injury is still found in some patients. Further follow-up is necessary.

Objective:To analyze the epidemiological and clinical characteristics of patients with imported corona virus disease 2019 (COVID-19) in Beijing City.Methods:A case-control study was performed to retrospectively analyze 69 cases of imported COVID-19 from abroad and 147 cases of domestic confirmed COVID-19 from China as a control group from January 20 to March 20, 2020 admitted to Beijing Ditan Hospital, Capital Medical University.The epidemiological and clinical characteristics were compared.Statistical analysis were performed by t test, Mann-Whitney U test, chi-square test and Fisher exact test. Results:The main sources of the cases in the import group were from the United Kingdom, Italy, Spain and other European countries, with 44.9%(31/69) of the overseas students entering the country by air. The age of the imported group (27(21, 40) years) was lower than the domestic group (43 (32, 59)years), the difference between the two groups was statistically significant ( U=2 828.500, P<0.01). Compared with the domestic group, the proportion of cases with contact history of confirmed cases in the imported group was lower (30.4%(21/69) vs 68.0%(100/147)), the interval between onset and admission ≤seven days was higher (81.2%(56/69) vs 66.0%(97/147)), the proportion of cases with underlying diseases was lower (21.7%(15/69) vs 44.2%(65/147)). The differences between the two groups were all statistically significant ( χ2=26.935, 5.233 and 10.175, respectively, all P<0.05). The proportion of mild cases in the imported group was higher than that in the domestic group (42.0%(29/69) vs 10.9%(16/147)). Seventeen cases with olfactory abnormality and 12 cases with taste abnormality were found in the imported group, while no olfactory and taste abnormality was found in the domestic group. The proportions of fever, weakness, muscle soreness and dyspnea were all lower than those of the domestic group, the differences between the two groups were all statistically significant ( χ2=13.851, 8.118, 9.730 and 16.255, respectively, all P<0.01). The proportions of cases with decreased lymphocyte absolute numbers (37.7%(26/69) vs 67.3%(99/147)) and increased C reactive protein level (15.9%(11/69) vs 51.8%(72/139)) were both lower than the domestic group, and the differences between the two groups were both statistically significant ( χ2=18.015 and 24.722, respectively, both P<0.01). The proportions of cases with ground glass shadow and consolidation of chest computed tomography were lower than those of the domestic group and the differences between the two groups were all statistically significant ( χ2=11.961 and 5.099, respectively, all P<0.05). In terms of complications, the proportions of cases with acute respiratory distress syndrome and acute myocardial injury were lower (2.9%(2/69) vs 10.9%(16/147) and 4.3%(3/69) vs 14.0%(16/114), respectively), and there were statistically significant differences between the two groups ( χ2=4.017 and 4.335, respectively, both P<0.05). There were no cases received mechanical ventilation and extracorporeal membrane oxygenation in the imported group, and the proportions of patients received oxygen therapy and antibiotic treatment were significantly lower than those in the domestic group (13.0%(9/69) vs 26.5%(39/147) and 13.0%(9/69) vs 39.5%(58/147), respectively) and the differences between the two groups were statistically significant ( χ2=4.942 and 15.797, respectively, both P<0.05). Conclusions:The majority of imported COVID-19 cases are mainly from European countries, mostly young and middle-aged, and mostly mild and ordinary types.The symptoms of olfactory and taste abnormality are found for the first time.

Objective:To learn about the iodine nutritional status of pregnant women in Suqian City, Jiangsu Province, and to provide evidence for scientific supplementation of iodine of pregnant women.Methods:From May 2016 to July 2020, five sampling districts were divided in each county (district) of Suqian City according to the oriation of east, west, south, north and center each year. One township (street) was selected from each district, and 20 pregnant women who lived in the local area for more than half a year were selected from each township (street). The 30 g of household salt samples of pregnant women and 5 ml of urine samples at random once were collected to test the salt iodine and urinary iodine content.Results:A total of 2 483 household salt samples of pregnant women were tested, and the median salt iodine was 23.9 mg/kg; among them, 2 454 were iodized salt, and the coverage rate of iodized salt was 98.8%; the qualified iodized salt was 2 383, the qualified rate of iodized salt was 97.1%, and the consumption rate of qualified iodized salt was 96.0%. There were statistically significant differences in coverage rate of iodized salt, qualified rate of iodized salt and consumption rate of qualified iodized salt between different years (χ 2 = 10.55, 13.23, 11.37, P < 0.05). A total of 2 483 urine samples of pregnant women were tested, and the median urinary iodine was 167.6 μg/L, which was at the appropriate iodine level. However, the median urinary iodine of pregnant women in 2020 was 146.7 μg/L, lower than the WHO/UNICEF/ICCIDD recommendation standard (150 μg/L). The differences of median urinary iodine of pregnant women in different years, pregnancy periods and regions were statistically significant ( H = 26.08, 8.17, 19.87, P < 0.05). Conclusions:The coverage rate of iodized salt, qualified rate of iodized salt and consumption rate of qualified iodized salt in Suqian City , meet the national standard for eliminating iodine deficiency disorders. Iodine nutrition of pregnant women in Suqian City is at an appropriate iodine level, but some pregnant women may have iodine deficiency.

Objective:To analyze the clinical characteristics of patients with acute glyphosate herbicide poisoning and the differences in the severity of poisoning.Methods:A retrospective analysis was performed on patients with acute glyphosate herbicide poisoning admitted to the First Affiliated Hospital of Zhengzhou University from January 2014 to December 2020. The general information, exposure time, poisoning dose, poisoning cause, poisoning route, clinical manifestations, laboratory examination results during hospitalization, treatment measures, hospital stays and prognosis of the patients were collected. The patients were graded according to the poisoning severity scoring standard of Chinese Expert Consensus on Diagnosis and Treatment of Acute Poisoning in 2016. The highest severity score during hospitalization was used as the final grade. According to the final grade, asymptomatic and mild patients were included in the mild group, and moderate, severe and death patients were included in the severe group. The independent sample T test or Mann-Whitney U test was used for measurement data, and χ2 test or Fisher's exact test was used for counting data. The differences of general data and clinical data between the two groups were compared. Results:According to the inclusion and exclusion criteria, 83 patients with acute glyphosate herbicide poisoning were selected as the study subjects. All patients survived, mainly mild poisoning (56.6%), with a male to female ratio of 33∶50, and an average age of 39 years. The number of poisoning cases increased yearly (the highest in 2019), and most cases occurred in spring and summer. The main cause of poisoning was suicide (71.1%), direct oral administration (83.1%) was the primary route of poisoning, and the dominating clinical manifestations were digestive symptoms (71.1%). Laboratory tests showed increased white blood cell count (WBC), neutrophil percentage (NEUT %) and D-dimer, and decreased hemoglobin and potassium. Compared with the mild group, patients in the severe group were older [(51±17) years vs. (35±19) years], had a higher proportion of suicide and direct oral administration, a longer hospital stay [8.0 (4.8, 12.0) d vs. 3.0 (2.0, 5.5) d], a higher dose of poisoning [200.0 (50.0, 200.0) mL vs. 30.0 (11.3, 57.5) mL], and higher NEUT % within 24 h of admission [(83.4±10.4) vs. (73.2±12.8)]. The increase of WBC, NEUT %, aspartate aminotransferase, prothrombin time, D-dimer and the decrease of serum potassium were more common in the severe group than the mild group, with statistical significance (all P<0.05). Conclusions:The number of patients with acute glyphosate herbicide poisoning is increasing yearly. Generally, the condition is mild and the prognosis is satisfying. The severity is more serious in the middle-aged and elderly patients andthose with direct oral administration, high toxic dose, and high NEUT % within 24 h of admission. Severe poisoning is more likely to cause changes in laboratory indicators.