[Similarities and differences of myocardial metabolic characteristics between HFpEF and HFrEF mice based on LC-MS/MS metabolomics].

Objective: To reveal the similarities and differences in myocardial metabolic characteristics between heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) mice using metabolomics. Methods: The experimental mice were divided into 4 groups, including control, HFpEF, sham and HFrEF groups (10 mice in each group). High fat diet and Nω-nitroarginine methyl ester hydrochloride (L-NAME) were applied to construct a"two-hit"HFpEF mouse model. Transverse aortic constriction (TAC) surgery was used to construct the HFrEF mouse model. The differential expression of metabolites in the myocardium of HFpEF and HFrEF mice was detected by untargeted metabolomics (UHPLC-QE-MS). Variable importance in projection>1 and P<0.05 were used as criteria to screen and classify the differentially expressed metabolites between the mice models. KEGG functional enrichment and pathway impact analysis demonstrated significantly altered metabolic pathways in both HFpEF and HFrEF mice. Results: One hundred and nine differentially expressed metabolites were detected in HFpEF mice, and 270 differentially expressed metabolites were detected in HFrEF mice. Compared with the control group, the most significantly changed metabolite in HFpEF mice was glycerophospholipids, while HFrEF mice presented with the largest proportion of carboxylic acids and their derivatives. KEGG enrichment and pathway impact analysis showed that the differentially expressed metabolites in HFpEF mice were mainly enriched in pathways such as biosynthesis of unsaturated fatty acids, ether lipid metabolism, amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, arachidonic acid metabolism and arginine and proline metabolism. The differentially expressed metabolites in HFrEF mice were mainly enriched in arginine and proline metabolism, glycine, serine and threonine metabolism, pantothenate and CoA biosynthesis, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and arachidonic acid metabolism, etc. Conclusions: HFpEF mice have a significantly different myocardial metabolite expression profile compared with HFrEF mice. In addition, biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, glycerophospholipid metabolism and arginine and proline metabolism are significantly altered in both HFpEF and HFrEF mice, suggesting that these metabolic pathways may play an important role in disease progression in both types of heart failure.

[Establishment of a mutation prediction model for evaluating the efficacy of immunotherapy in renal carcinoma].

OBJECTIVE: To establish a mutation prediction model for efficacy assessment, the genomic sequencing data of renal cancer patients from the MSKCC (Memorial Sloan Kettering Cancer Center) pan-cancer immunotherapy cohort was used. METHODS: The genomic sequencing data of 121 clear cell renal cell carcinoma patients treated with immune checkpoint inhibitors (ICI) in the MSKCC pan-cancer immunotherapy cohort were obtained from cBioPortal database (http://www.cbioportal.org/) and they were analyzed by univariate and multivariate Cox regression analysis to identify mutated genes associated with ICI treatment efficacy, and we constructed a comprehensive prediction model for drug efficacy of ICI based on mutated genes using nomogram. Survival analysis and time-dependent receiver operator characteristic curves were performed to assess the prognostic value of the model. Transcriptome and genomic sequencing data of 538 renal cell carcinoma patients were obtained from the TCGA database (https://portal.gdc.cancer.gov/). Gene set enrichment analysis was used to identify the potential functions of the mutated genes enrolled in the nomogram. RESULTS: We used multivariate Cox regression analysis and identified mutations in PBRM1 and ARID1A were associated with treatment outcomes in the patients with renal cancer in the MSKCC pan-cancer immunotherapy cohort. Based on this, we established an efficacy prediction model including age, gender, treatment type, tumor mutational burden (TMB), PBRM1 and ARID1A mutation status (HR=4.33, 95%CI: 1.42-13.23, P=0.01, 1-year survival AUC=0.700, 2-year survival AUC=0.825, 3-year survival AUC=0.776). The validation (HR=2.72, 95%CI: 1.12-6.64, P=0.027, 1-year survival AUC=0.694, 2-year survival AUC=0.709, 3-year survival AUC=0.609) and combination (HR=2.20, 95%CI: 1.14-4.26, P=0.019, 1-year survival AUC=0.613, 2-year survival AUC=0.687, 3-year survival AUC=0.526) sets confirmed these results. Gene set enrichment analysis indicated that PBRM1 was involved in positive regulation of epithelial cell differentiation, regulation of the T cell differentiation and regulation of humoral immune response. In addition, ARID1A was involved in regulation of the T cell activation, positive regulation of T cell mediated cyto-toxicity and positive regulation of immune effector process. CONCLUSION: PBRM1 and ARID1A mutations can be used as potential biomarkers for the evaluation of renal cancer immunotherapy efficacy. The efficacy prediction model established based on the mutation status of the above two genes can be used to screen renal cancer patients who are more suitable for ICI immunotherapy.

[Application value of non-invasive ventilation combined with high flow nasal cannula oxygen therapy in sequential treatment of patients with chronic obstructive pulmonary disease after mechanical ventilation].

Objective: To investigate the value of non-invasive ventilation (NIV) combined with high flow nasal cannula oxygen therapy (HFNCO) in sequential treatment of patients with chronic obstructive pulmonary disease after mechanical ventilation. Methods: Chronic obstructive pulmonary disease with acute exacerbation (AECOPD) patients with invasive mechanical ventilation (MV) and successful withdrawal admitted into Huxi Affiliated Hospital of Jining Medical College from January 2018 to December 2019 were enrolled for perspective study. The patients were divided into treatment group (n=40) and control group (n=33) by random number table method. The treatment group was given NIV and HFNCO, the control group was given NIV treatment alone. Bedside ultrasound was used to measure the patients' diaphragmatic motion, and the differences between the two groups of patients before treatment, 24, 48 and 72 h after treatment were compared in diaphragmatic excursions during quiet breathing (DEq), diaphragmatic excursions during deep breathing(DEd), diaphragmatic shallow fast breathing index (D-RSBI), arterial oxygen partial pressure (PaO(2)), arterial partial pressure of carbon dioxide (PaCO(2)), re-tracheal intubation rate, mortality rate for 28 days and average duration of NPPV treatment within 3 days. Results: There were no statistically significant differences in DEq, DEd, D-RSBI, PaO(2) and PaCO(2) between the two groups before treatment (all P>0.05). After 24 h treatment, DEd decreased in both groups, D-RSBI increased in both groups, However, D-RSBI [(1.33±0.56) vs (1.62±0.59) times·min(-1)·mm(-1)] in the treatment group was significantly lower than the control group, P=0.034. After 72 h treatment, DEd [(41.4±8.1) vs (37.8±6.0) mm] was significantly higher than the control group, D-RSBI [(1.02±0.27) vs (1.22±0.43) times·min(-1)·mm(-1)] was significantly lower than the control group (all P<0.05). The average duration of NIV treatment time [(7.5±1.2) vs (9.3±2.6) h] in the treatment group was significantly shorter than that in the control group (P<0.01). There were no statistically significant differences in PaO(2), PCO(2), re-tracheal intubation rate and the mortality rate of 28 days. Conclusion: NIV combined with HFNCO sequential therapy can effectively relieve diaphragm fatigue and promote recovery of respiratory muscle strength, and it's better than NIV alone.

[The therapeutic experience of blunt pancreatic trauma].

Objective: To summarize the experience of treatment for blunt pancreatic trauma. Methods: The clinical data of 52 patients with blunt pancreatic trauma admitted to the Department of Pancreatic and Biliary Surgery of the First Affiliated Hospital of Harbin Medical University from January 2013 to June 2018 were analyzed retrospectively.There were 40 male and 12 female patients, aging from 12 to 112 years with a median age of 35.5 years.According to the organ injury scale by American Association for the Surgery of Trauma(AAST) for pancreatic injury severity, 15 cases were in grade Ⅰ(28.8%), 20 cases were in grade Ⅱ(38.5%), 10 cases were in grade Ⅲ(19.2%),5 cases were in grade Ⅳ(9.6%) and 2 cases were in grade Ⅴ(3.8%). Isolated blunt pancreatic trauma occurred in 11(21.2%) patients including 5 cases of grade Ⅰ,5 cases of grade Ⅱ and 1 case of grade Ⅲ, and associated injuries existed in 41 patients(78.8%). Results: Among 52 patients, 36 patients(69.2%) were transferred from other hospitals and 16(30.8%) patients were admitted through the emergency department. Finally, 49 patients(94.2%) were cured and 3 patients (5.8%) died.For the 15 cases of grade Ⅰ,9 patients were managed non-operatively, 5 cases underwent peritoneal lavage and drainage after surgery for the other injured abdominal organs, and 1 patient received percutaneous catheter drainage(PCD) with non-operative treatment. For the 20 cases of grade Ⅱ,4 cases only received non-operative treatment and 2 cases also received PCD. Besides, 2 cases underwent debridement and drainage for peripancreatic necrotic tissue and external drainage for pancreatic pseudocyst retrospectively after about 25 days of getting injured. As for patients who received exploratory laparotomy, 5 patients underwent suture repair associated with external drainage, and 7 patients were managed only with external drainage. For the 10 cases of grade Ⅲ,6 patients were cured through distal pancreatectomy and splenectomy with external drainage, while 2 patients underwent endoscopic retrograde cholangiopancreatography and ductal stenting, and the other 2 patients just received debridement and drainage for peripancreatic necrotic tissue.For the 5 cases of grade Ⅳ,2 patients underwent jejunostomy and abdominal cavity drainage, 1 patient had a pancreaticoduodenectomy with drainage,1 patient received suture repair of the pancreas and pancreaticojejunostomy, and 1 patient was managed with suture repair of the head of pancreas and external drainage.For the 2 patients of grade Ⅴ,1 patient received exploratory laparotomy and gauze compression packing hemostasis, and the other patient underwent pancreaticoduodenal repair, gastrointestinal anastomosis, duodenal exclusion surgery and external drainage. Conclusion: According to the AAST classifications, associated injuries, physiological status and intraoperative situation, it could be better to make a comprehensive judgment, achieve early diagnosis and take appropriate individualized treatment strategy, and to improve the overall therapeutic effect for blunt pancreatic trauma.

[Transurethral resection of prostate treatment for recurrence of a multilocular prostatic cystadenoma: a case report].

Multilocular prostatic cystadenoma (MPC) is a rare benign tumor that originates from the prostate itself. MPC is usually characterized by large multilocular cysts located between the rectum and bladder. The clinical presentation includes obstructive voiding symptoms, such as poor stream, intermittency, sensation of incomplete emptying, acute urinary retention and sometimes constipation symptoms due to mechanical compression of the lower intestine. Most of the previously reported patients with MPC underwent open surgery. Although the natural history of MPC remains unknown, surgical excision may not always be necessary. Here we report the case of a 49-year-old male, treated by transurethral electroresection of prostate (TURP) for prostate cyst one and half years before．His biopsy of TURP showed benign prostatic tissue with no evidence of malignancy. However, the symptoms of urinary tract obstruction were obviously aggravated after the operation. Acute urinary retention occurred intermittently 3 times. In our hospital, his total prostate specific antigen (tPSA) was 5.440 µg/L, free prostate specific antigen (fPSA) was 1.528 µg/L. After examination, it was considered as benign lesions clearly. In the operation of TURP, we found that the tumor was multilocular cystic. Histologically, the cell was mucus. Concerning the immunophenotype, CK5/6(+) , p40(+), PSA(+), P504S(+), PAX-2(-), PAX-8(-), MUC1(+), MUC5ac(+), the results of special staining were as follows: AB(+), PAS(+). At the end of the follow up 3 months later, the routine semen analysis results showed that his semen volume was 3 mL and the sperm density and sperm mobility were normal. At the end of the follow up eight months later, the patient remained free of lower urinary tract symptoms and there were no signs of recurrence. His international prostate symptom score (I-PSS) had dropped from 32 to 4, and quality of life score (QOL) had dropped from 6 to 2. MPC is a rare benign tumor originating from the prostate. TURP may aggravate the symptoms of lower urinary tract obstruction in patients with MPC, and may be temporarily observed for some asymptomatic young and middle-aged patients.

MiR-4299 suppresses non-small cell lung cancer cell proliferation, migration and invasion through modulating PTEN/AKT/PI3K pathway.

OBJECTIVE: The aim of the present study was to investigate clinical significances and biological roles of miR-4299 in non-small cell lung cancer (NSCLC) PATIENTS AND METHODS: Expression of miR-4299 in NSCLC tissues and matched non-tumor tissues was determined by quantitative real-time PCR (qRT-PCR). The correlations between miR-4299 expression and clinicopathological characteristics and prognosis were also analyzed. MTT assay and Transwell assay were performed to determine the proliferation, migration and invasion. Western blotting was used to examine the expressing patterns of PTEN/AKT/PI3K signaling pathway-related proteins. RESULTS: We found that the expression level of miR-4299 was downregulated in NSCLC tissues and cell lines. Low miR-4299 expression was positively correlated with TNM stage (p=0.002), histological grade (p=0.002) and lymph node metastasis (p=0.028). Moreover, Kaplan-Meier survival analysis showed that the patients with low miR-4299 expression had shorter survival time than those with high miR-4299 expression (p=0.0011). More importantly, multivariate analysis suggested that decreased miR-4299 expression was a poor independent prognostic predictor for NSCLC patients (p=0.009). Functionally, overexpression of miR-4299 inhibited the proliferation, migration and invasion in A549 cells. Mechanistically, the results of Western blot showed that miR-4299 exhibited its tumor-suppressive role by modulating PTEN/AKT/PI3K signaling pathway. CONCLUSIONS: We firstly indicated that miR-4299 may be a candidate independent marker for NSCLC prognosis and suppressed the progression of NSCLC by modulating the activation of PTEN/AKT/PI3K signaling pathway, suggesting that miR-4299 could be a potential target for developing therapies in treating NSCLC.

Evaluation of the application of high volume hemofiltration in sepsis combined with acute kidney injury.

OBJECTIVE: To investigate the safety and effectiveness of the application of high-volume hemofiltration (HVHF) in children with sepsis combined with acute kidney injury. PATIENTS AND METHODS: A total of 76 child patients were enrolled and randomly divided equally (n=38) into control and the observation groups respectively. The control group received conventional volume hemofiltration (the ultrafiltration rate of 35-50 ml/kg/h), and the observation group received HVHF (50-100 ml/kg/h). RESULTS: The serum Interleukin-6 (IL-6), Tumor Necrosis Factor-a (TNF-α) and creatinine levels were significantly lower in the observation group than the control group at 6 h, 12 h, 24 h and 48 h of hemofiltration (p<0.05). The efficacy rate of treatment was improved. The mortality rate and incidence rate of complications were decreased, and the treatment course was significantly shortened (p<0.05). CONCLUSIONS: The application of HVHF in children with sepsis combined with acute kidney injury has a better safety and effectiveness.

Correction to: Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains.

There were errors in Fig. 1 of the originally published article. Correct fig. 1 is presented here.

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

OBJECTIVE: To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. METHODS: The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. RESULTS: CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. CONCLUSION: Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

[Application of flurbiprofen axetil in pain management associated during transrectal ultrasound-guided prostate biopsy].

OBJECTIVE: To examine the effects of perioperative intravenous administration of flurbiprofen axetil (FA) on pain associated with transrectal ultrasound-guided prostate biopsy. METHODS: This was a randomized,controlled study. Eighty-one patients who underwent 12 core prostate biopsy were included in the study. The patients were randomly assigned to one of three groups (n=27 in each) by type of procedure during prostate biopsy. Group intrarectal local anesthesia (IRLA) received intrarectal 5% (0.05 g/L) lidocaine gel 60 mg, 5 minutes before the procedure alone; Group FA received intravenous flurbiprofen axetil (1 mg/kg) 1 hour before the procedure; Group IRLA+FA received intrarectal 5% lidocaine gel 60 mg, 5 minutes before the procedure and intravenous flurbiprofen axetil (1 mg/kg) 1 hour before the procedure. The patients were asked to score the pain by using visual analogue scale (VAS) in 4 situations,including when the probe was inserted (VASI),during anesthesia (VAS II),during biopsy (VAS III) and 20 minutes after biopsy (VAS IV). The findings were evaluated with analysis of variance,and the Tukey post hoc test was followed with an overall 2-tailed significance level at α =0.05. P1, P value between Group IRLA and Group FA; P2, P value between Group FA and Group IRLA +FA, P3, P value between Group IRLA and Group IRLA +FA. The bonferroni method was used to adjust the test level, α=0.017,a P value of less than 0.017 was accepted as the threshold for statistical significance. RESULTS: No major complications,including sepsis and severe rectal bleeding,were noted in any patient. There were no differences in general condition of the patients before procedure among the 3 groups. There were statistically significant differences in VAS scores among the 3 groups in VAS II (5.7±2.2, 3.0±1.5,3.3±1.9,respectively,P=0.012) and VAS III (6.7±2.3,3.0±2.1,2.9±1.6,respectively,P=0.001). There were no differences in the pain scores among the 3 groups during probe insertion (VAS I, 3.2±1.0,4.1±2.1,4.2±1.7, respectively,P=5.752) and 20 minutes after biopsy (VAS IV, 1.4±2.1,1.0±0.9,1.1±0.7,respectively,P=3.772). Between-column differences among the 3 groups were VAS II (P1=0.007,P2=5.655,P3=0.001,respectively) and VAS III(P1=0.008,P2=7.517,P3=0.001,respectively),the differences between Group IRLA and Group FA,Group IRLA and Group IRLA +FA in VAS II and VAS III were statistically significant. CONCLUSION: The intravenous flurbiprofen axetil was found to be more effective than intrarectal lidocaine gel alone.

Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains.

Blastocystis is one common protist inhabiting in gastrointestinal tracts of animals and humans. Examining the subtypes has important implications for assessing the zoonotic potential of Blastocystis and intestinal health of hosts. In the present study, a total of 497 fecal samples collected from 37 wild animal species in Qinling Mountains were investigated for the presence and subtypes of Blastocystis. Of them, 200 (40.2%) were positive for Blastocystis and 13 subtypes were found, including eight known subtypes (STs1-3, 5, 10, 12-14) and five possible novel subtypes (temporarily named as STs18-22), with ST10 as the predominate subtype and the subtype ST5 was detected in an ostrich for the first time. These findings indicated the wide distribution and specific subtype characteristics of Blastocystis in wild animals of Qinling Mountains.

Variants of ubiquitin-specific peptidase 24 play a crucial role in lung cancer malignancy.

Ubiquitin is a critical modifier regulating the degradation and function of its target proteins during posttranslational modification. Here we found that ubiquitin-specific peptidase 24 (USP24) is highly expressed in cell lines with enhanced malignancy and in late-stage lung cancer clinical samples. Studying single-nucleotide polymorphisms (SNPs) of USP24 using genomic DNA of lung cancer patients revealed an increase in SNP 7656C/T. When using RNA specimens instead of the genomic DNA of lung cancer patients, we found significant increases in the ratios of variants 930C/T and 7656T/C, suggesting that variants at these two sites are not only caused by the SNP of DNA but also by the RNA editing. USP24-930T and USP24-7656C increase USP24 expression levels by increasing RNA stability. Knocking down USP24 increased Suv39h1 level through a decrease in mouse double-minute 2 homolog levels, thus enhancing lysine-9 methylation of histone H3, and resulting in the prevention of lung cancer malignancy. In conclusion, as USP24 variant analysis revealed a higher ratio of variants in blood specimens of lung cancer patients than that in normal individuals, USP24-930T and USP24-7656C might be useful as diagnostic markers for cancer detection.

Pleural mesothelial cells promote expansion of IL-17-producing CD8+ T cells in tuberculous pleural effusion.

IL-17-producing CD8(+) T lymphocytes (Tc17 cells) have recently been detected in many cancers and autoimmune diseases. However, the possible implication of Tc17 cells in tuberculous pleural effusion remains unclarified. In this study, distribution and phenotypic features of Tc17 cells in both tuberculous pleural effusion (TPE) and peripheral blood from patients with tuberculosis were determined. The effects of proinflammatory cytokines and local accessory cells (pleural mesothelial cells) on Tc17 cell expansion were also explored. We found that TPE contained more Tc17 cells than the blood. Compared with IFN-γ-producing CD8(+) T cells, Tc17 cells displayed higher expression of chemokine receptors (CCRs) and lower expression of cytotoxic molecules. In particularly, Tc17 cells in TPE exhibited high expression levels of CCR6, which could migrate in response to CCL20. Furthermore, IL-1ß, IL-6, IL-23, or their various combinations could promote Tc17 cell expansion from CD8(+) T cells, whereas the proliferative response of Tc17 cells to above cytokines was lower than that of Th17 cells. Pleural mesothelial cells (PMCs) were able to stimulate Tc17 cell expansion via cell contact in an IL-1ß/IL-6/IL-23 independent fashion. Thus this study demonstrates that Tc17 cells marks a subset of non-cytotoxic, CCR6(+) CD8(+) T lymphocytes with low proliferative capacity. The overrepresentation of Tc17 cells in TPE may be due to Tc17 cell expansion stimulated by pleural proinflammatory cytokines and to recruitment of Tc17 cells from peripheral blood. Additionally, PMCs may promote the production of IL-17 by CD8(+) T cells at sites of TPE via cell-cell interactions.

Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

Disruption of glucose homeostasis and induction of insulin resistance by elevated free fatty acids in human L02 hepatocytes.

Free fatty acids (FFA) have been implicated as an important causative link between obesity, insulin resistance, and Type 2 diabetes. However, the underlying mechanisms especially for FFA-mediated hepatic insulin resistance are not fully elucidated. Here, we investigated the impaired sites in insulin signaling pathways and mechanisms of insulin resistance induced by elevated FFA in L02 hepatocytes. L02 cells were cultured in Dulbecco's modified eagle medium containing various concentrations of palmitic acid (PA) for 24 h followed by 10(-7) mol/l insulin stimulation. In some experiments, cells were pre-treated with enzymatic inhibitor Wortmannin (10(-6) mol/l). Glucose levels in medium, cytosolic glycogen contents, and phosphoenolpyruvate carboxykinase (PEPCK) activity were measured. Protein level of insulin receptor substrate (IRS)-2 and phosphorylated Akt were detected by Western blot analysis. L02 cells treated with high levels of PA exhibited increased glucose levels, whereas hepatic glycogen contents were decreased in a dose-dependent manner as compared to the control cells. There was a significant attenuation of IRS- 2 protein expression in the cells cultured with PA, and Wortmannin intervention exhibited different IRS-2 protein level with or without PA treatment. In accordance with the reduced IRS-2 level, the insulin-stimulated phosphorylation of Akt was diminished in the PA-treated cells. Basal PEPCK activity and insulin- regulated PEPCK activity were overstimulated in the cells incubated with PA. These data indicate high levels of FFA can disrupt glucose homeostasis, inflict some defects in insulin signaling, and induce insulin resistance in L02 cells.

Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain.

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the alpha-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na2HPO4, 0.03% KH2PO4, 0.02% MgCl2, 0.3% CaCl2, 0.25% Na2CO3, 0.1% glucose, and 20 microg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40 degrees C, respectively. This enzyme was stable over a pH range of 8-11. Its residual activity remained at 100% when treated under a temperature of less than 45 degrees C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60 degrees C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.