Construction and validation of somatic mutation-derived long non-coding RNAs signatures of genomic instability to predict prognosis of hepatocellular carcinoma.

BACKGROUND: Long non-coding RNAs (LncRNAs) have been found to be a potential prognostic factor for cancers, including hepatocellular carcinoma (HCC). Some LncRNAs have been confirmed as potential indicators to quantify genomic instability (GI). Nevertheless, GI-LncRNAs remain largely unexplored. This study established a GI-derived LncRNA signature (GILncSig) that can predict the prognosis of HCC patients. AIM: To establish a GILncSig that can predict the prognosis of HCC patients. METHODS: Identification of GI-LncRNAs was conducted by combining LncRNA expression and somatic mutation profiles. The GI-LncRNAs were then analyzed for functional enrichment. The GILncSig was established in the training set by Cox regression analysis, and its predictive ability was verified in the testing set and TCGA set. In addition, we explored the effects of the GILncSig and TP53 on prognosis. RESULTS: A total of 88 GI-LncRNAs were found, and functional enrichment analysis showed that their functions were mainly involved in small molecule metabolism and GI. The GILncSig was constructed by 5 LncRNAs (miR210HG, AC016735.1, AC116351.1, AC010643.1, LUCAT1). In the training set, the prognosis of high-risk patients was significantly worse than that of low-risk patients, and similar results were verified in the testing set and TCGA set. Multivariate Cox regression analysis and stratified analysis confirmed that the GILncSig could be used as an independent prognostic factor. Receiver operating characteristic curve analysis of the GILncSig showed that the area under the curve (0.773) was higher than the two LncRNA signatures published recently. Furthermore, the GILncSig may have a better predictive performance than TP53 mutation status alone. CONCLUSION: We established a GILncSig that can predict the prognosis of HCC patients, which will help to guide prognostic evaluation and treatment decisions.

Ligamentum teres hepatis as a graft for portal and/or superior mesenteric vein reconstruction: From bench to bedside.

BACKGROUND: Pancreaticoduodenectomy combined with portal vein (PV) and/or superior mesenteric vein (SMV) resection in patients with pancreaticobiliary malignancy has become a common surgical procedure. There are various grafts currently used for PV and/or SMV reconstruction, but each of these grafts have certain limitations. Therefore, it is necessary to explore novel grafts that have an extensive resource pool, are low cost with good clinical application, and are without immune response rejection or additional damage to patients. AIM: To observe the anatomical and histological characteristics of the ligamentum teres hepatis (LTH) and evaluate PV/SMV reconstruction using an autologous LTH graft in pancreaticobiliary malignancy patients. METHODS: In 107 patients, the post-dilated length and diameter in resected LTH specimens were measured. The general structure of the LTH specimens was observed by hematoxylin and eosin (HE) staining. Collagen fibers (CFs), elastic fibers (EFs), and smooth muscle (SM) were visualized by Verhoeff-Van Gieson staining, and the expression of CD34, factor VIII-related antigen (FVIIIAg), endothelial nitric oxide synthase (eNOS), and tissue type plasminogen activator (t-PA) were detected using immunohistochemistry in LTH and PV (control) endothelial cells. PV and/or SMV reconstruction using the autologous LTH was conducted in 26 patients with pancreaticobiliary malignancies, and the outcomes were retrospectively analyzed. RESULTS: The post-dilated length of LTH was 9.67 ± 1.43 cm, and the diameter at a pressure of 30 cm H2O was 12.82 ± 1.32 mm at the cranial end and 7.06 ± 1.88 mm at the caudal end. Residual cavities with smooth tunica intima covered by endothelial cells were found in HE-stained LTH specimens. The relative amounts of EFs, CFs and SM in the LTH were similar to those in the PV [EF (%): 11.23 ± 3.40 vs 11.57 ± 2.80, P = 0.62; CF (%): 33.51 ± 7.71 vs 32.11 ± 4.82, P = 0.33; SM (%): 15.61 ± 5.26 vs 16.74 ± 4.83, P = 0.32]. CD34, FVIIIAg, eNOS, and t-PA were expressed in both LTH and PV endothelial cells. The PV and/or SMV reconstructions were successfully completed in all patients. The overall morbidity and mortality rates were 38.46% and 7.69%, respectively. There were no graft-related complications. The postoperative vein stenosis rates at 2 wk, 1 mo, 3 mo and 1 year were 7.69%, 11.54%, 15.38% and 19.23%, respectively. In all 5 patients affected, the degree of vascular stenosis was less than half of the reconstructed vein lumen diameter (mild stenosis), and the vessels remained patent. CONCLUSION: The anatomical and histological characteristics of LTH were similar to the PV and SMV. As such, the LTH can be used as an autologous graft for PV and/or SMV reconstruction in pancreaticobiliary malignancy patients who require PV and/or SMV resection.

Heat shock factor 1 promotes neurite outgrowth and suppresses inflammation in the severed spinal cord of geckos.

The low intrinsic growth capacity of neurons and an injury-induced inhibitory milieu are major contributors to the failure of sensory and motor functional recovery following spinal cord injury. Heat shock transcription factor 1 (HSF1), a master regulator of the heat shock response, plays neurogenetic and neuroprotective roles in the damaged or diseased central nervous system. However, the underlying mechanism has not been fully elucidated. In the present study, we used a gecko model of spontaneous nerve regeneration to investigate the potential roles of gecko HSF1 (gHSF1) in the regulation of neurite outgrowth and inflammatory inhibition of macrophages following spinal cord injury. gHSF1 expression in neurons and microglia at the lesion site increased dramatically immediately after tail amputation. gHSF1 overexpression in gecko primary neurons significantly promoted axonal growth by suppressing the expression of suppressor of cytokine signaling-3, and facilitated neuronal survival via activation of the mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases and phosphatidylinositol 3-kinase/protein kinase B pathways. Furthermore, gHSF1 efficiently inhibited the macrophage-mediated inflammatory response by inactivating IkappaB-alpha/NF-kappaB signaling. Our findings show that HSF1 plays dual roles in promoting axonal regrowth and inhibiting leukocyte inflammation, and provide new avenues of investigation for promoting spinal cord injury repair in mammals.

Improvement of inflammatory response and gastrointestinal function in perioperative of cholelithiasis by Modified Xiao-Cheng-Qi decoction.

BACKGROUND: In the perioperative period of biliary surgery, various factors can induce the release of a large number of inflammatory factors, leading to an imbalance in pro-inflammatory and anti-inflammatory responses and resulting in gastrointestinal (GI) dysfunction. Enhanced Recovery After Surgery protocols in biliary surgery have been shown to reduce the stress response and accelerate postoperative recovery. It is crucial to reduce the inflammatory response and promote the recovery of GI function after biliary surgery, both of which are the basis and key for perioperative care and postoperative recovery. AIM: To better understand the effects of Modified Xiao-Cheng-Qi decoction (MXD) on inflammatory response and GI function in the perioperative management of cholelithiasis and their correlation. METHODS: This was a prospective randomized placebo-controlled trial, in which 162 patients who received biliary tract surgery were randomly assigned to three groups: MXD group, XD group, and placebo-control group. The observed parameters included frequency of bowel sounds, time of first flatus and defecation, time of diet, and amount of activity after surgery. The serum levels of C-reactive protein (CRP), interleukin (IL)-6, IL-10, serum amyloid A protein (SAA), and substance P were measured by the enzyme-linked immunosorbent assay. Then, the spearman correlation coefficient was used to analyze the relationship between the indicators of GI function and inflammation. RESULTS: Compared to the placebo-control, improvements in GI function were observed in the MXD groups including reduced incidence of nausea, vomiting, and bloating; and earlier first exhaust time, first defecation time, and feeding time after surgery (P < 0.05). On the 1st and 2nd d after surgery, IL-6, CRP and SAA levels in MXD group were lower than that in placebo control, but substance P level was higher, compared to the control (P < 0.05). Functional diarrhea occurred in both MXD and XD groups without any other adverse effects, toxic reactions, and allergic reactions. Diarrhea was relieved after the discontinuation of the investigational remedies. Bowel sounds at 12 h after surgery, the occurring time of the first flatus, first defecation, postoperative liquid diet and semi-liquid diet were significantly correlated with levels of IL-6, CRP, SAA and substance P on second day after surgery (P < 0.05). CONCLUSION: Treatment with MXD can relieve inflammatory response and improve GI function after surgery. Moreover, there are significant correlations between them. Furthermore, it does not cause serious adverse reactions.

[Observation of effect of Wu Yun Liu Qi on reproductive hormones in varicocele patients].

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

Global and Regional Trends and Projections of Infective Endocarditis-Associated Disease Burden and Attributable Risk Factors from 1990 to 2030.

Objective To forecast the future burden and its attributable risk factors of infective endocarditis (IE). Method We analyzed the disease burden of IE and its risk factors from 1990 to 2019 using the Global Burden of Disease 2019 database and projected the disease burden from 2020 to 2030 using a Bayesian age-period-cohort model. Results By 2030, the incidence of IE will increase uncontrollably on a global scale, with developed countries having the largest number of cases and developing countries experiencing the fastest growth. The affected population will be predominantly males, but the gender gap will narrow. The elderly in high-income countries will bear the greatest burden, with a gradual shift to middle-income countries. The incidence of IE in countries with middle/high-middle social-demographic indicators (SDI) will surpass that of high SDI countries. In China, the incidence rate and the number of IE will reach 18.07 per 100,000 and 451,596 in 2030, respectively. IE-associated deaths and heart failure will continue to impose a significant burden on society, the burden on women will increase and surpass that on men, and the elderly in high-SDI countries will bear the heaviest burden. High systolic blood pressure has become the primary risk factor for IE-related death. Conclusions This study provides comprehensive analyses of the disease burden and risk factors of IE worldwide over the next decade. The IE-associated incidence will increase in the future and the death and heart failure burden will not be appropriately controlled. Gender, age, regional, and country heterogeneity should be taken seriously to facilitate in making effective strategies for lowering the IE disease burden.

Association Between Lipid Profiles and Left Ventricular Hypertrophy: New Evidence from a Retrospective Study.

Objective To explore the association between lipid profiles and left ventricular hypertrophy in a Chinese general population. Methods We conducted a retrospective observational study to investigate the relationship between lipid markers [including triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL-cholesterol, apolipoprotein A-I, apolipoprotein B, lipoprotein[a], and composite lipid profiles] and left ventricular hypertrophy. A total of 309,400 participants of two populations (one from Beijing and another from nationwide) who underwent physical examinations at different health management centers between 2009 and 2018 in China were included in the cross-sectional study. 7,475 participants who had multiple physical examinations and initially did not have left ventricular hypertrophy constituted a longitudinal cohort to analyze the association between lipid markers and the new-onset of left ventricular hypertrophy. Left ventricular hypertrophy was measured by echocardiography and defined as an end-diastolic thickness of the interventricular septum or left ventricle posterior wall > 11 mm. The Logistic regression model was used in the cross-sectional study. Coxmodel and Coxmodel with restricted cubic splines were used in the longitudinal cohort. Results In the cross-sectional study, for participants in the highest tertile of each lipid marker compared to the respective lowest, triglycerides [odds ratio (OR): 1.250, 95%CI: 1.060 to 1.474], HDL-cholesterol (OR: 0.780, 95%CI: 0.662 to 0.918), and lipoprotein(a) (OR: 1.311, 95%CI: 1.115 to 1.541) had an association with left ventricular hypertrophy. In the longitudinal cohort, for participants in the highest tertile of each lipid marker at the baseline compared to the respective lowest, triglycerides [hazard ratio (HR): 3.277, 95%CI: 1.720 to 6.244], HDL-cholesterol (HR: 0.516, 95%CI: 0.283 to 0.940), non-HDL-cholesterol (HR: 2.309, 95%CI: 1.296 to 4.112), apolipoprotein B (HR: 2.244, 95%CI: 1.251 to 4.032) showed an association with new-onset left ventricular hypertrophy. In the Coxmodel with forward stepwise selection, triglycerides were the only lipid markers entered into the final model. Conclusion Lipids levels, especially triglycerides, are associated with left ventricular hypertrophy. Controlling triglycerides level potentiate to be a strategy in harnessing cardiac remodeling but deserve to be further investigated.

Incidentally discovered asymptomatic splenic hamartoma misdiagnosed as an aneurysm: A case report.

BACKGROUND: Splenic hamartoma (SH) is a rare, benign vascular proliferation that is often found incidentally. It may be misdiagnosed as a splenic aneurysm or splenic malignancy. CASE SUMMARY: A 21-year-old male patient was admitted to our hospital with a complaint of an incidentally discovered asymptomatic splenic space-occupying lesion for 2 wk. Abdominal computed tomography (CT) scan showed a circular low-density shadow in the hilum of the spleen. Contrast-enhanced CT revealed an aneurysm located in the hilum of the spleen before operation. Laparoscopic splenectomy was performed and postoperative pathology revealed the presence of SH. CONCLUSION: Imaging studies are insufficient for the differential diagnosis of SH from other diseases, and laparoscopic splenectomy is a less invasive procedure and useful for the diagnostic purpose as well.

Establishment of a Faba Bean Banker Plant System with Predator Orius strigicollis for the Control of Thrips Dendrothrips minowai on Tea Plants under Laboratory Conditions.

The stick tea thrip Dendrothrips minowai (Priesner) (Thysanoptera: Thripidae) is a destructive pest in tea plantations in south and southwest China. To control this pest, a non-crop banker plant system was developed using a polyphagous predator Orius strigicollis (Poppius) (Heteroptera: Anthocoridae) with the black bean aphid Aphis fabae (Scopoli) (Hemiptera: Aphididae) as an alternative prey and the faba bean Vicia faba as the banker plant to support the predator in targeting the pest. The fitness of A. fabae on tea plants and faba bean was evaluated to determine its host specificity. Moreover, the control efficacy of the banker plant system on D. minowai on tea plants was tested in the laboratory and compared with that of direct release of O. strigicollis. The experiments showed that faba bean was an excellent non-crop host for A. fabae because, while the aphid population increased quickly on faba bean, it could only survive for up to 9 days on tea plants. Compared with direct release of O. strigicollis, lower densities of pest were observed when introducing the banker plant system. Our results indicate that this banker plant system has the potential to be implemented in the field to improve the control of the pest thrips.

[Effect of bilateral microsurgical subinguinal varicocelectomy on asthenozoospermia and oligozoospermia: A report of 109 cases].

OBJECTIVE: To investigate the effects of bilateral microsurgical subinguinal varicocelectomy (BMSV) in patients with asthenozoospermia or oligozoospermia. METHODS: Totally 147 patients with male infertility received BMSV from January 2018 to May 2019, of whom 109 had complete data recorded. We retrospectively analyzed the clinical data, including the total sperm count per ejeculate, sperm concentration and sperm motility before and after surgery, and the rate of natural conception achieved during the follow-up. RESULTS: BMSV achieved a total effectiveness rate of 79.00% in improving the percentage of progressively motile sperm (a rise of ≥20%) and a marked effectiveness rate of 70.00% (a rise of ≥50%) in the 100 cases of asthenozoospermia as compared with the baseline, with a mean recovery time of (110.13 ± 37.43) days. Besides, a total effectiveness rate of 74.29% (an increase of ≥20%) and a marked effectiveness rate of 71.43% (an increase of ≥50%) were attained in the improvement of sperm concentration in the 35 cases of oligozoospermia, with a mean recovery time of (117.00 ± 48.79 ) days. A natural conception rate of 30.30% was observed during the follow-up. No severe adverse events occurred postoperatively. CONCLUSIONS: BMSV is significantly effective for the treatment of asthenozoospermia and oligozoospermia.

[HPV infection of the external genitalia in men whose female partners have cervical HPV infection].

OBJECTIVE: Human papilloma virus (HPV) is a necessary cause of cervical cancer and is also closely related to penile cancer, oropharyngeal cancer, and anal cancer in males. However, few studies are reported on male HPV. This study aimed to investigate HPV infection of the external genitalia in men whose female partners have cervical HPV infection. METHODS: We collected the relevant data on the male outpatients whose partners had cervical HPV infection in our Department of Urology and Andrology from August to December 2016. We obtained samples with nylon swabs from the glans penis, corona, inner layer of the prepuce and penile body and detected different types of HPV infection using the Hybribio HPV typing kit, PCR and membrane hybridization. RESULTS: Valid data were collected from 140 males, which showed 83.5% of HPV infection of the external genitalia, including 60 cases of HPV6 (43.2%), 27 cases of HPV16 (19.4%), 14 cases of HPV39 (10.1%), 13 cases of HPV18 (9.4%), 13 cases of HPV58 (9.4%), and 13 cases of HPV52 (9.4%). Redundant prepuce was found in 75.5% of the males, but there was no statistically significant difference in the incidence rate of HPV infection between the normal and redundant prepuce groups (P > 0.05). CONCLUSIONS: Men who have the female partners with positive cervical HPV are at high risk of HPV infection and therefore need to be screened and treated so as to reduce HPV infection in both sexes.

[Relationship of abnormal sperm DNA methylation with early spontaneous abortion].

OBJECTIVE: To investigate the relationship between the abnormal sperm DNA methylation level and early spontaneous abortion. METHODS: We randomly selected 98 males who met the inclusion criteria and whose wives suffered from unexplained abortion or embryo abortion, and included another 46 normal healthy men present for pre-pregnancy check-up as controls. We examined the semen quality and sperm morphology, obtained the sperm DNA fragmentation index (DFI) by modified sperm chromatin dispersion, and measured the sperm DNA methylation level using the methylated DNA quantification kit and the colorimetric method. RESULTS: Compared with the normal controls, the men in the unexplained abortion group showed a significantly lower rate of big-halo sperm (ï¼»45.50 ± 26.27ï¼½ vs ï¼»36.49 ± 23.06ï¼½%, P = 0.038), a higher rate of abnormal-head sperm (ï¼»77.08± 12.21ï¼½ vs ï¼»81.09± 10.89ï¼½%, P = 0.049), and a lower level of sperm DNA methylation (ï¼»0.47 ± 0.33ï¼½ vs ï¼»0.36 ± 0.26ï¼½ ng/µl, P = 0.035). The sperm DNA methylation level was positively correlated with the percentage of big-halo sperm (OR=0.546, P<0.01). Multivariate regression analysis manifested that sperm head abnormality was an independent risk factor of early spontaneous abortion or embryo abortion (OR=1.032, P = 0.049), while the high methylation level was protective factor against early spontaneous abortion or embryo abortion (OR=0.244, P = 0.03). CONCLUSIONS: The abnormal level of sperm DNA methylation may be one of the important reasons for early spontaneous abortion or embryo abortion.

[Semen quality and its influencing factors in preconception males in Nanjing area].

OBJECTIVE: To investigate the semen quality and its influencing factors in preconception males in Nanjing area so as to provide some evidence for working out effective intervention measures. METHODS: Totally 687 men receiving preconceptional physical examination were enrolled in this study. A questionnaire survey was conducted among the subjects along with an analysis of their semen quality. RESULTS: The median of sperm concentration was 63.3 x 10(6)/ml (95% CI [19.88-119] x 10(6)/ml). The median of grade a sperm was 33.03% (95% CI [19.38-55.05]%), that of grade a + b sperm was 52.08% (95% CI [39.53-69.37]%), and that of teratosperm was 91.75% (95% CI [69-100]%). The median concentration of seminal plasma PMN-elastase was 195.55 ng/ml (95% CI [76.16-3330.38] ng/ml) and that of seminal plasma zinc was 7.62 µmol/L (95% CI [1.5-23, 45] µmol/L). The positive rates of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Gardnerella vaginalis (GV) were 42.4%, 0.3%, and 2.4%, respectively. The median of sperm DNA fragmentation index (DFI) of those whose wives had a history of adverse pregnancy was 20.25% (95% CI [2.15-68.25]%). Multivariate logistic regression analysis suggested that mental stress (OR 1.567, 95% CI [1.081-2.27]) and sedentariness (OR 1.772, 95% CI [1.211-2.592]) were independent risk factors for asthenospermia. CONCLUSION: The sperm quality of preconception males in Nanjing area is not encouraging, and it can be improved by changing undesirable lifestyle and reducing mental stress.

[Treatment of varicocele by microsurgical varicocelectomy: report of 94 cases].

OBJECTIVE: To investigate the clinical effect of microsurgical varicocelectomy in the treatment of varicocele. METHODS: We retrospectively analyzed the clinical data of 94 varicocele patients treated by microsurgical varicocelectomy, of whom 36 complained of testicular pain or dragging and distending discomfort, and 58 infertility or oligoasthenospermia. Microsurgical varicocelectomy was performed through inguinal or subinguinal approach. Complications were observed and semen parameters detected through follow-up visits at 1, 3 and 6 months after the operation. RESULTS: Testicular pain or discomfort disappeared in 21 (65.6%) of the 32 followed-up patients who had complained of such symptoms, was relieved in 6 (18.8%), and remained unimproved in the other 5 (15.6%). The 56 followed-up patients with infertility or oligoasthenospermia all showed significantly improved sperm concentration and motility (grade a + b sperm), (15.47 +/- 3.21) x 10(6)/ml and (13.34 +/- 5.16)% at 3 months, and (18.39 +/- 4.05) x 10(6)/ml and (17.23 +/- 4.69)% at 6 months after operation, as compared with (8.26 +/- 1.68) x 10(6)/ml and (5.25 +/- 1.09)% preoperatively (P < 0.01). CONCLUSION: Microsurgical varicocelectomy can effectively improve symptoms and semen parameters in varicocele patients.

[Preparation of carbon nanoparticle paclitaxel suspension and pharmacokinetic study of intraperitoneal chemotherapy].

OBJECTIVE: To prepare carbon nanoparticle-paclitaxel suspension(CNPS) and to study the pharmacokinetics of intraperitoneal chemotherapy with CNPS. METHODS: Saturated absorption capacity of carbon nanoparticle suspension (CNS) and paclitaxel were detected by high performance liquid chromatography in order to prepare the above suspension. Wistar rats were randomly divided into the experimental group (A) and the control group (B), to which intraperitoneal injections of CNPS and paclitaxel were given respectively. At different time points, measure the blood samples, mesenteric lymph nodes, and intraperitoneal lavage fluid were collected to measure the concentration of paclitaxel. RESULTS: One ml CNS could absorb 7 mg paclitaxel by maximum. The ratio of area under the curve (AUC) in the plasma of group A to group B was 0.63. The ratio of AUC in lymph nodes of group A to group B was 0.75 and that in intraperitoneal lavage fluid was 1.25. The metabolic half-life (t1/2) of paclitaxel in the plasma of group A was 1.61 times as long as that of group B. The t1/2 of paclitaxel in intraperitoneal lavage fluid of group A was 0.88 as long as that of Group B. The t1/2 of paclitaxel in lymph nodes of group A was 1.10 as long as that of Group B. CONCLUSIONS: CNS has a high absorption capacity with paclitaxel. Intraperitoneal chemotherapy by CNPS is characterized by low drug concentration in the blood, high drug concentration in the peritoneal cavity and high safety. However, the targeting and lymphatic retention effect are not significant. The mechanism warrants further investigation.

[Protective functions of recombinant protein targeted at RANKL against hepatic ischemia/reperfusion injury transfected by retrovirus in mice].

OBJECTIVE: To explore the protective functions of recombinant protein RANK-Fc against hepatic ischemia/reperfusion injury and clarify its possible mechanism. METHODS: Sixty male Balb/c mice were randomly divided into 3 groups according to different treatments: serum-free medium control (Sham) group, target gene retrovirus (RANK-Fc) group and empty vector retrovirus (eGFP) group. All mice were injected with 2.5 ml solution (with or without retrovirus) within 6 seconds via tail vein. After 3 days, the model of 70% hepatic ischemia/reperfusion was induced under warm conditions for 90 minutes after different periods of reperfusion in RANK-Fc and eGFP groups; Sham group underwent the same procedure without the occlusion of blood supply. Blood and liver samples were obtained at different time points (1, 3, 6 and 24 h; n = 5 in each). Reverse transcription-polymerase chain reaction (RT-PCR) was used for the evaluation of eGFP mRNA expression. RANK-Fc was assessed by Western blot. Liver transaminases and histopathological changes were used for the evaluation of hepatic injury. The activity of NF-κB in liver nucleus was analyzed by Western blot and immunohistochemistry. The activation level of JNK was also assessed by Western blot. Liver homogenate levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The differences between three treatment groups at each time point were detected by the one-way ANOVA. Statistical analysis for inter-comparison was performed by Student's t test. RESULTS: RANK-Fc and eGFP were successfully expressed in liver after hydrodynamics-based transfection. Compared with eGFP group, RANK-Fc significantly improved liver functions at the same time point (P < 0.01), decreased NF-κB nuclear translocation (t = 6.726, P < 0.01)and JNK phosphorylation (t = 6.713, P < 0.01)and obviously suppressed the release of pro-inflammatory cytokine TNF-α (t = 4.779, P < 0.01) and IL-6 (t = 5.482, P < 0.01). Morphological injuries were markedly alleviated while the expressions of immunohistochemical positive cells and TUNEL staining positive cells decreased in RANF-Fc group. CONCLUSION: RANK-Fc has protective functions against hepatic ischemia/reperfusion injury in mice. Its mechanism is at least partially related with the suppressions of proinflammatory NF-κB and proapoptotic JNK signaling pathways.

[Metabolic flux analysis of L-valine fermentation in Corynebacterium glutamicum].

In industrial fermentation of amino acids the cells are often forced to synthesize the biochemicals excessive of their physiological needs. The knowledge of metabolic networks and their regulation relevant usually come from biochemical research, especially from enzymology, not from engineering study. To enrich the knowledge of metabolic sub-network of L-valine syntheses for higher production of L-valine, Corynebacterium glutamicum AS1.495 and its genetic derivatives AA361, AAT231, AATV341 were used for metabolic flux analysis. AS1.495 is a leucine auxotrophic (Leu-), and the three derivatives carry additional mutations. AA361 contains D-aspartic acid-beta-hydroxamate supersensitive marker (Leu-, L-AAHss), AAT231 (Leu-, L-AAHss, 2-TAr) is D-aspartic acid-beta-hydroxamate supersensitive and 2-thiazole alanine resistant, and AAT341 (Leu-, L-AAHss, 2-TAr, Vd-) is a D-aspartic acid-beta-hydroxamate supersensitive, 2-thiazole alanine resistant and valine-decompose-ability imperfect (Vd-). The concentrations of extra-cellular metabolites were determined under sub-steady-state of the batch culture. The metabolic flux distribution maps of the four strains were obtained, compared and analyzed. Our analysis showed that the flux ratio of EMP and HMP from the glucose-6-phosphate had increased from 0.205 in the parental strain AS1.495 to 0.321 in the multiple-mutation strain AATV341; the flux ratio of L-valine synthesis branch and the rest branches from the pyruvate node increased from 0.188 in AS1.495 to 3.29 in AATV341; the flux of lactic acid synthesis branch decreased from 11.1 in AS1.495 to 1.16 in AATV341; the flux of L-valine synthesis branch increased from 5.37 in AS1.495 to 37.3 in AATV341; and the productivity of L-valine correspondently increased from 4 g/L in AS1.495 to 24.5 g/L in AATV341. These results indicate that the introduction of analog supersensitive marker L-AAH55 and/or analog resistant marker 2-TAr skew the metabolic flux towards the formation of L-valine. This study revealed the usefulness of the metabolic flux analysis as a tool for verification of existing production strains. The analysis may play an important role in helping us b to rationally re-design metabolism for further improvement of fermentation process.