[Efficacy and safety of various doses of hybutimibe monotherapy or in combination with atorvastatin for primary hypercholesterolemia: a multicenter, randomized, double-blind, double-dummy, parallel-controlled phase â ¢ clinical trial].

Objective: To evaluate the efficacy and safety of hybutimibe monotherapy or in combination with atorvastatin in the treatment of primary hypercholesterolemia. Methods: This was a multicenter, randomized, double-blind, double-dummy, parallel-controlled phase Ⅲ clinical trial of patients with untreated primary hypercholesterolemia from 41 centers in China between August 2015 and April 2019. Patients were randomly assigned, at a ratio of 1∶1∶1∶1∶1∶1, to the atorvastatin 10 mg group (group A), hybutimibe 20 mg group (group B), hybutimibe 20 mg plus atorvastatin 10 mg group (group C), hybutimibe 10 mg group (group D), hybutimibe 10 mg plus atorvastatin 10 mg group (group E), and placebo group (group F). After a dietary run-in period for at least 4 weeks, all patients were administered orally once a day according to their groups. The treatment period was 12 weeks after the first dose of the study drug, and efficacy and safety were evaluated at weeks 2, 4, 8, and 12. After the treatment period, patients voluntarily entered the long-term safety evaluation period and continued the assigned treatment (those in group F were randomly assigned to group B or D), with 40 weeks' observation. The primary endpoint was the percent change in low density lipoprotein cholesterol (LDL-C) from baseline at week 12. Secondary endpoints included the percent changes in high density lipoprotein cholesterol (HDL-C), triglyceride (TG), apolipoprotein B (Apo B) at week 12 and changes of the four above-mentioned lipid indicators at weeks 18, 24, 38, and 52. Safety was evaluated during the whole treatment period. Results: Totally, 727 patients were included in the treatment period with a mean age of (55.0±9.3) years old, including 253 males. No statistical differences were observed among the groups in demographics, comorbidities, and baseline blood lipid levels. At week 12, the percent changes in LDL-C were significantly different among groups A to F (all P<0.01). Compared to atorvastatin alone, hybutimibe combined with atorvastatin could further improve LDL-C, TG, and Apo B (all P<0.05). Furthermore, there was no significant difference in percent changes in LDL-C at week 12 between group C and group E (P=0.991 7). During the long-term evaluation period, there were intergroup statistical differences in changes of LDL-C, TG and Apo B at 18, 24, 38, and 52 weeks from baseline among the statins group (group A), hybutimibe group (groups B, D, and F), and combination group (groups C and E) (all P<0.01), with the best effect observed in the combination group. The incidence of adverse events was 64.2% in the statins group, 61.7% in the hybutimibe group, and 71.0% in the combination group during the long-term evaluation period. No treatment-related serious adverse events or adverse events leading to death occurred during the 52-week study period. Conclusions: Hybutimibe combined with atorvastatin showed confirmatory efficacy in patients with untreated primary hypercholesterolemia, which could further enhance the efficacy on the basis of atorvastatin monotherapy, with a good overall safety profile.

[Research progress on cow's milk protein allergy in preterm infant].

Parenteral nutrition plays an important role in the early growth and development of preterm infants. Milk protein is the main protein source for enteral nutrition in preterm infants. Although the incidence of cow's milk protein allergy (CMPA) in preterm infants is relatively low, the symptoms are atypical and easily confused with other diseases, leading to incorrect diagnosis and treatment measures, and seriously affecting the growth, development and prognosis of preterm infants. This article reviews the epidemiology, clinical manifestations, auxiliary examination, diagnosis and differential diagnosis, treatment and prevention of CMPA in premature infants. The onset of CMPA in premature infants was late. Few patients had rectal bleeding, and bile-like vomiting and symptoms similar to necrotizing enterocolitis were more common. The diagnosis is mainly based on the avoidance test. For children diagnosed with CMPA, they should be avoided through the mother's diet or replaced with deep hydrolyzed formula milk or amino acid formula milk. However, in-depth clinical and basic research is still needed on how to identify CMPA in preterm infants early.

[Changes of immunohistochemical biomarkers before and after neoadjuvant chemotherapy in breast cancer and their prognosis].

Objective: To investigate changes in the expression of immunohistochemical (IHC) markers and factors associated with the effect of chemotherapy before and after neoadjuvant chemotherapy (NAC). Methods: A retrospective study included 200 breast cancer patients treated with NAC between January 2016 and December 2018. We analyzed the changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 in pre- and post-treated samples and the predictive factors of NAC. Results: Among the 200 cases, 16 cases were luminal A, 108 cases were luminal B, 36 cases were HER2+subtype, and 40 cases were basal-like. Twenty-five patients (12.50%) achieved pathological complete remission (PCR).There were significant differences in PR and Ki-67 before and after NAC but there were no differences in ER and HER2.In univariate analysis, factors associated with PCR were tumor less than 5 cm(P=0.009), non-luminal breast cancer (P=0.001), ER negative(P=0.001), PR negative (P=0.029) and HER2 positive(P=0.001). Tumor less than 5 cm [P=0.020, OR=2.581, 95%CI (1.207, 5.753)], ER negative [P=0.011, OR=2.264, 95%CI (1.207, 4.248)] and HER2 positive[P=0.007, OR=2.412, 95%CI (1.275, 4.561)] remained predictive variables in multivariate analysis after correction for the other variables. Conclusions: The expression of Ki-67 decreases after NAC. Negative PR and ER and positive HER2 status are related to the efficacy of pCR for breast cancer, and have guiding significance for the prognosis evaluation of NAC.

[Research advances on the characteristics and wound healing promoting effect of in-situ forming injectable hydrogels].

Research of in-situ induced repair and regeneration is a multi- and inter-disciplinary field, which is of important potentials in the treatment of both large-area deep burns and chronic wounds such as diabetic skin ulcers. In-situ forming injectable hydrogels which are hydrogel-like biomaterials that can spontaneously gelatinize in physiological condition when applied in local wounds have been explored in recent years. This kind of biomaterials contain extracellular matrix, in which cells promoting wound repairing can be added if required, and can work as release-controlled carriers for active peptides such as growth factors to simulate local wound microenvironment and induce the repair and regeneration. Herein, characteristics and function of promoting wound repair and regeneration about in-situ forming injectable hydrogels were reviewed, including material types and their relevant working mechanisms, advantages, existing problems, etc.

[Efficacy and safety of early initiation of sacubitril-valsartan therapy in patients with acute decompensated heart failure].

Objective: To assess the efficacy and safety of the initiation of sacubitril-valsartan （ARNI） therapy, as compared with ACEI therapy, after hemodynamic stabilization among patients hospitalized for acute decompensated heart failure （ADHF）. Methods: A total of 199 hospitalized patients for ADHF in our department from January 2017 to June 2019 were included in this retrospective analysis. According to the medication early after hemodynamic stabilization, patients were divided into ARNI group (n=92) and ACEI group (n=107). Among the included patients, 61 patients with newly diagnosed heart failure at the time of admission were also divided into ARNI group (n=30) and ACEI group (n=31) according to the applied medication. Clinical baseline data and follow-up results of enrolled patients were collected through the electronic medical records at admission, outpatient and telephone follow-up. The primary effectiveness observation index was left ventricular ejection fraction (LVEF) and left ventricular end diastolic dimension (LVEDD) measured by echocardiography; the secondary observation index was death from any causes and hospitalization for heart failure. Safety outcomes were the incidences of symptomatic hypotension, worsening renal function, hyperkalemia, and angioedema. Results: The clinical baseline characteristics were similar between ARNI group and ACEI group（all P>0.05）. The duration of follow up was (15.2±6.5) months in all patients enrolled, (12.3±5.0) months in ARNI group, and (18.2±6.5) months in ACEI group. At the end of follow-up, prevalence of an absolute LVEF increase of more than 5% was 48.9% (45/92) in ANRI group and 25.2% (27/107) in ACEI group (P=0.001). Percent of LVEF increase to more than 50% was 17.4% (16/92) in ANRI group and 3.7% (4/107) in ACEI group (P=0.001). Percent of patients with more than 10 mm LVEDD reduction was 14.1% (13/92) in ANRI group and 3.7% (4/107) in ACEI group (P=0.009). All-cause mortality rate was 5.7% (5/88) in ARNI group and 15.3% (13/85) in ACEI group (P=0.038). Rate of re-hospitalization due to heart failure was 50% (46/92) in ARNI group and 71% (76/107) in ACEI group（P=0.002）.The rates of symptomatic hypotension, worsening renal function, hyperkalemia, and angioedema were similar between ARNI group and ACEI group (all P>0.05). In patients with first diagnosed heart failure，percent of LVEF increase to more than 50% was 30% (9/30) in ANRI group and 6.5% (2/31) in ACEI group (P=0.017). Percent of more than 10 mm LVEDD reduction was 26.7%(8/30) in ANRI group and 3.2%(1/31) in ACEI group (P=0.012). Percent of an absolute LVEF increase of more than 5% was 53.3% (16/30) in ANRI group and 51.6% (16/31) in ACEI group (P=0.893). Re-hospitalization due to heart failure was 23.3% (7/30） in ARNI group and 73.3% (11/31) in ACEI group（P<0.01）. Rate of all-cause death tended to be lower in patients receiving ARNI (3.4% (1/29)) as compared to patients receiving ACEI (13.0% (3/23), P=0.197）. Conclusions: Among patients with heart failure with reduced ejection fraction hospitalized for ADHF, the initiation of ARNI therapy after hemodynamic stabilization is associated with a more significant improvement of cardiac remodeling and pump function than ACEI therapy and satisfactory safety. In ADHF patients with first diagnosed heart failure, initiation of ARNI therapy after hemodynamic stabilization can more effectively improve cardiac remodeling and pump function than treatment with ACEI.

[Clinical study of cell sheets containing allogeneic keratinocytes and fibroblasts for the treatment of partial-thickness burn wounds].

Objective: To evaluate the efficacy and safety of cell sheets containing allogeneic keratinocytes and fibroblasts in the treatment of partial-thickness burn wounds. Methods: The cell sheets containing allogeneic keratinocytes and fibroblasts were constructed using polyurethane biofilm as carrier. Then gross observation and histological observation were conducted. From April 2016 to December 2017, Changhai Hospital of Naval Medical University recruited patients with acute partial-thickness burn wounds that met the inclusion criteria for this prospective and positively self-controlled clinical trial. Recruitment of 40 acute partial-thickness burn wounds were planned with each selected single wound being not smaller than 10 cm×10 cm and not more than 5% total body surface area (TBSA). Each wound was equally divided into two areas, which were recruited into cell sheet group and conventional treatment group according to the random number table. The wounds in cell sheet group were covered by cell sheet and then sterile gauze as secondary dressings. Depending on the wound healing and exudation, the sterile gauze was replaced every 1 to 3 day (s) after the treatment was started, and the cell sheet was replaced every 7 days (namely dressing changing). The wounds in conventional treatment group were covered by sulfadiazine silver cream gauze and then dressed with sterile gauze, with the dressings changed every 2 to 3 days depending on wound exudation. On treatment day 5, 7, 10, and 14, the wound healing rates in the two groups were calculated. The complete wound healing time, the total number of dressing changes, and the status of wound infection during treatment were recorded. The Visual Analogue Scale was used to score the pain at the first dressing change. Scar formation of patients was followed up for 6 to 12 months after injury. Safety indicators including vital signs, laboratory examination indexes, and adverse reactions during treatment were observed. Data were statistically analysed with Wilcoxon rank sum test and Bonferroni correction. Results: (1) Each prepared cell sheet had a diameter of about 8 cm and was about 49 cm(2) in size, containing 2 or 3 layers of keratinocytes and fibroblasts. (2) A total of 43 patients were enrolled, of whom 3 patients dropped out of the study. Of the 40 patients who completed the treatment, there were 22 males and 18 females who were aged 1 to 57 year (s), with total burn area of 2% to 26% TBSA. (3) On treatment day 5, 7, 10, and 14, the wound healing rates in cell sheet group were significantly higher than those in conventional treatment group (Z=4.205, 4.258, 3.495, 2.521, P<0.05 or P<0.01). The complete wound healing time in cell sheet group was 7 (6, 8) days, which was significantly shorter than 11 (7, 14) days in conventional treatment group (Z=4.219, P<0.01). The total number of wound dressing changes in cell sheet group was 1 (1, 2) times, which was significantly less than 6 (4, 7) times in conventional treatment group (Z=5.464, P<0.01). (4) The wounds in cell sheet group in 31 patients healed before the first dressing change. The pain score of wounds in the first dressing change in cell sheet group of 9 patients was 1 (0, 1) point, while the pain score of wounds in the first dressing change in conventional treatment group of 40 patients was 2 (1, 3) points. There was no obvious infection in the wounds in both groups of 40 patients before the wound healing. Nine patients completed the follow-up after the trial. In 6 patients, no scar formation was observed in cell sheet group or conventional treatment group. The color of wounds in cell sheet group was consistent with normal skin, and there was only a small amount of pigment deposition in the wounds of conventional treatment group. Three patients developed pigment deposition only in the wounds of cell sheet group but obvious scars in conventional treatment group. (5) The abnormal fluctuations of vital signs including body temperature, blood pressure, heart rate, respiratory rate, and laboratory examination indexes of all patients during treatment were alleviated through the process of burn wound healing. No obvious adverse reactions or abnormalities related to the treatment were observed. Conclusions: The cell sheet containing allogeneic keratinocytes and fibroblasts can reduce the number of dressing changes, accelerate wound epithelialization, shorten wound healing time, reduce pain during dressing change in the treatment of partial-thickness burn wounds, and it may reduce scar hyperplasia after wound healing because of accelerating wound epithelization. Its clinical application is simple, safe, and effective.

[Preliminary effect observation on the application of micro-negative pressure in children with small-area deep partial-thickness burn].

Objective: To preliminarily observe the effects of application of micro-negative pressure in children with small-area deep partial-thickness burn. Methods: From January 2016 to August 2018, 64 children with small-area deep partial-thickness burn who were admitted to the Department of Burn Surgery of the First Affiliated Hospital of Naval Medical University were recruited in this prospective randomized controlled study. According to the random number table, they were divided into negative pressure group [18 boys and 14 girls, aged (3.9±1.6) years with total burn area of (5.5±2.2)% total body surface area (TBSA)] and conventional group [20 boys and 12 girls, aged (3.8±1.7) years with total burn area of (5.8±1.6)% TBSA], with 32 patients in each group. After admission, simple debridement was performed in the patients of 2 groups. After that, the children in negative pressure group were treated with micro-negative pressure with negative pressure material replaced every 3 to 5 days. Children in conventional group were treated with silver sulfadiazine cream with dressing change every other day. On post injury day (PID) 14 and 21, general wound observation was performed, the wound healing rate was calculated, the exudates from the wounds were cultured and the positive detection rate was calculated. The number of patients requiring surgical skin grafting was recorded and the rate of surgical skin grafting was calculated, and the complete wound healing time was recorded in the patients of 2 groups. Scar formation was evaluated by the Vancouver Scar Scale (VSS) in 3, 6, and 12 months after wound healing. Data were processed with chi-square test, t test, Bonferroni correction, and analysis of variance for repeated measurement. Results: (1) On PID 14, all the necrotic tissue in the wounds of patients in negative pressure group was removed, with few exudates, and most of the wounds had been epithelialized; most of necrotic tissue in the wounds of patients in conventional group was removed, with more exudates and smaller wound healing area than those in negative pressure group. On PID 21, most of the wounds of patients in negative pressure group were healed, and the exudates were rare, while the wound healing area of patients in conventional group was significantly smaller than that in negative pressure group with more exudates. (2) On PID 14 and 21, the wound healing rates [(49.8±3.3)% and (95.8±2.4)%] of patients in negative pressure group were significantly higher than those in conventional group [(40.0±3.2)% and (75.3±2.5)%, t=11.899, 33.461, P<0.01]. (3) On PID 14 and 21, the positive detection rates of wound bacteria of patients in negative pressure group were significantly lower than those in conventional group (χ(2)=6.275, 5.741, P<0.05). (4) The rate of surgical skin grafting of patients in negative pressure group was significantly lower than that in conventional group (χ(2)=5.333, P<0.05). (5) The complete wound healing time of patients in negative pressure group [(23.9±2.3) d] was significantly shorter than that in conventional group [(27.9±1.8) d, t=-7.806, P<0.01]. (6) In 3, 6, and 12 months after wound healing, the VSS scores [(6.9±1.8), (5.6±1.4), (3.4±1.5) points] of patients in negative pressure group were significantly lower than those in conventional group [(9.0±1.5), (7.4±2.0), (5.7±1.6) points, t=-4.987, -4.127, -5.988, P<0.01]. Conclusions: In comparison with routine dressing change, the treatment of application of micro-negative pressure in children with small-area deep partial-thickness burn can significantly improve the wound healing rate and rate of surgical skin grafting, decrease the wound infection rate, shorten the wound healing time, and improve the wound healing quality.

Differentiation between malignant and benign breast masses: combination of semi-quantitative analysis on DCE-MRI and histogram analysis of ADC maps.

AIM: To investigate the performance of combined semi-quantitative analysis on dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) and histogram analysis of diffusion-weighted imaging (DWI) for distinguishing malignant from benign breast masses. MATERIALS AND METHODS: This study included 178 patients with breast masses (benign:malignant=88:9) who underwent both DCE-MRI and DWI. The semi-quantitative parameters, derived from DCE-MRI, included maximum slope of increase (MSI), signal intensity slope (SIslope), initial percentage of enhancement (Einitial), percentage of peak enhancement (Epeak), early signal enhancement ratio (ESER), and second enhancement percentage (SEP). Histogram parameters derived from apparent diffusion coefficient (ADC) maps included ADCmin, ADCmax, ADCmean, ADC10, ADC25, ADC50, ADC75, ADC90, skewness, and kurtosis. All parameters were compared between malignant and benign groups, and their differences were tested using independent-samples t-test or Mann-Whitney test. Receiver operating characteristic (ROC) curves were used to determine the diagnostic value of each significant parameter. RESULTS: Among semi-quantitative parameters, SIslope exhibited the best diagnostic performance in predicting malignancy (cut-off value, 0.096; ROC, 0.756; sensitivity, 86.7%; specificity, 61.4%). Among histogram parameters, ADC10 exhibited the best diagnostic performance in predicting malignancy (cut-off value, 1.051; ROC, 0.885; sensitivity, 86.7%; specificity, 84.1%). The optimal diagnostic performance of combined ADC10 and SIslope (area under curve [AUC], 0.888; sensitivity, 82.2%; specificity, 95.5%) was significantly better than SIslope alone (p<0.001). Moreover, the combination showed higher AUC (0.888 versus 0.885) than ADC10 alone, but the difference was not statistically significant (p=0.914). CONCLUSION: SIslope and ADC10 are significant predictors for breast malignancy. The combination of DCE-MRI and DWI improves differentiating performance.

Overexpression of a glycine-rich protein gene in Lablab purpureus improves abiotic stress tolerance.

Glycine-rich protein (GRP) is involved in the response to abiotic and biotic stresses in plants. A novel GRP gene in Lablab purpureus has been identified. The cDNA of LpGRP was obtained from an SSH library constructed with root tissues of L. purpureus MEIDOU 2012 by waterholding for 10 days. The function of LpGRP was also evaluated in Arabidopsis. The cDNA of LpGRP has 555 bp and encodes a 184-amino acid protein. LpGRP was induced by drought and improved tolerance to abiotic stress. In LpGRP overexpressing Arabidopsis, the tolerance of transgenic seedlings to drought and salt was improved, and transgenic seeds showed insensitivity to both ABA and NaCl. The insensitivity to ABA indicated that there was crosstalk between LpGRP and ABA-responsive genes. These results indicated that LpGRP is a drought-responsive gene that can increase the drought and salt tolerance of Arabidopsis seedlings overexpressing LpGRP.

HOI-02 induces apoptosis and G2-M arrest in esophageal cancer mediated by ROS.

Reactive oxygen species (ROS) are chemically reactive molecules that perform essential functions in living organisms. Accumulating evidence suggests that many types of cancer cells exhibit elevated levels of ROS. Conversely, generation of ROS has become an effective method to kill cancer cells. (E)-3-hydroxy-3-(4-(4-nitrophenyl)-2-oxobut-3-en-1-yl) indolin-2-one, which is an NO2 group-containing compound designated herein as HOI-02, generated ROS and, in a dose-dependent manner, decreased esophageal cancer cell viability and inhibited anchorage-independent growth, followed by apoptosis and G2-M arrest. Moreover, results of an in vivo study using a patient-derived xenograft mouse model showed that HOI-02 treatment suppressed the growth of esophageal tumors, without affecting the body weight of mice. The expression of Ki-67 was significantly decreased with HOI-02 treatment. In addition, the phosphorylation of c-Jun, and expression of p21, cleaved caspase 3, and DCFH-DA were increased in the HOI-02-treated group compared with the untreated control group. In contrast, treatment of cells with (E)-3-(4-(4-aminophenyl)-2-oxobut-3-en-1-yl)-3-hydroxyindolin-2-one, which is an NH2 group-containing compound designated herein as HOI-11, had no effect. Overall, we identified HOI-02 as an effective NO2 group-containing compound that was an effective therapeutic or preventive agent against esophageal cancer cell growth.

Whole genome and transcriptome sequencing of matched primary and peritoneal metastatic gastric carcinoma.

Gastric cancer is one of the most aggressive cancers and is the second leading cause of cancer death worldwide. Approximately 40% of global gastric cancer cases occur in China, with peritoneal metastasis being the prevalent form of recurrence and metastasis in advanced disease. Currently, there are limited clinical approaches for predicting and treatment of peritoneal metastasis, resulting in a 6-month average survival time. By comprehensive genome analysis will uncover the pathogenesis of peritoneal metastasis. Here we describe a comprehensive whole-genome and transcriptome sequencing analysis of one advanced gastric cancer case, including non-cancerous mucosa, primary cancer and matched peritoneal metastatic cancer. The peripheral blood is used as normal control. We identified 27 mutated genes, of which 19 genes are reported in COSMIC database (ZNF208, CRNN, ATXN3, DCTN1, RP1L1, PRB4, PRB1, MUC4, HS6ST3, MUC17, JAM2, ITGAD, IREB2, IQUB, CORO1B, CCDC121, AKAP2, ACAN and ACADL), and eight genes have not previously been described in gastric cancer (CCDC178, ARMC4, TUBB6, PLIN4, PKLR, PDZD2, DMBT1and DAB1).Additionally,GPX4 and MPND in 19q13.3-13.4 region, is characterized as a novel fusion-gene. This study disclosed novel biological markers and tumorigenic pathways that would predict gastric cancer occurring peritoneal metastasis.

Empirical Selection of Informative Microsatellite Markers within Co-ancestry Pig Populations Is Required for Improving the Individual Assignment Efficiency.

The Lanyu is a miniature pig breed indigenous to Lanyu Island, Taiwan. It is distantly related to Asian and European pig breeds. It has been inbred to generate two breeds and crossed with Landrace and Duroc to produce two hybrids for laboratory use. Selecting sets of informative genetic markers to track the genetic qualities of laboratory animals and stud stock is an important function of genetic databases. For more than two decades, Lanyu derived breeds of common ancestry and crossbreeds have been used to examine the effectiveness of genetic marker selection and optimal approaches for individual assignment. In this paper, these pigs and the following breeds: Berkshire, Duroc, Landrace and Yorkshire, Meishan and Taoyuan, TLRI Black Pig No. 1, and Kaohsiung Animal Propagation Station Black pig are studied to build a genetic reference database. Nineteen microsatellite markers (loci) provide information on genetic variation and differentiation among studied breeds. High differentiation index (FST) and Cavalli-Sforza chord distances give genetic differentiation among breeds, including Lanyu's inbred populations. Inbreeding values (FIS) show that Lanyu and its derived inbred breeds have significant loss of heterozygosity. Individual assignment testing of 352 animals was done with different numbers of microsatellite markers in this study. The testing assigned 99% of the animals successfully into their correct reference populations based on 9 to 14 markers ranking D-scores, allelic number, expected heterozygosity (HE) or FST, respectively. All miss-assigned individuals came from close lineage Lanyu breeds. To improve individual assignment among close lineage breeds, microsatellite markers selected from Lanyu populations with high polymorphic, heterozygosity, FST and D-scores were used. Only 6 to 8 markers ranking HE, FST or allelic number were required to obtain 99% assignment accuracy. This result suggests empirical examination of assignment-error rates is required if discernible levels of co-ancestry exist. In the reference group, optimum assignment accuracy was achievable achieved through a combination of different markers by ranking the heterozygosity, FST and allelic number of close lineage populations.

Suppression of tumor angiogenesis by targeting the protein neddylation pathway.

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin-RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.

Characterization of hTERT-immortalized caprine mammary epithelial cells.

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and ß-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock.

Abundantly expressed hepatic genes and their differential expression in liver of prelaying and laying geese.

Geese have a short egg-laying period and a low egg production rate. To induce and maintain egg laying, genes related to generating hepatic lipid for yolk deposition should be adequately expressed. Liver mRNA from 6 laying geese was extracted and used for construction of a full-length enriched cDNA library. About 2,400 clones containing gene sequences were determined and National Center for Biotechnology Information Gallus gallus Gene Index databases were used to compare and analyze these sequences. Ten highly expressed genes were selected to determine the differential expression between laying and prelay goose liver. Tissue distribution data showed that very low density apolipoprotein II, liver type fatty acid binding protein, vitellogenin I, and vitellogenin II transcripts were specifically expressed in the liver of laying geese. Ovoinhibitor, preproalbumin, alpha-2-hs-glycoprotein, and vitamin D binding protein mRNA were highly expressed in the liver and to a lesser extent in other tissues. Ovotransferrin mRNA was expressed in liver, ovary, oviduct, shell gland, brain, and adipose tissues. The concentration of transthyretin mRNA was high in the liver and brain. The mRNA concentrations of liver type fatty acid binding protein, alpha-2-hs-glycoprotein, and transthyretin in the livers of laying and prelay geese were not different. The concentrations of hepatic ovotransferrin, ovoinhibitor, preproalbumin, very low density apolipoprotein II, vitellogenin I, vitellogenin II, and vitamin D binding protein mRNA were higher in the liver of laying geese than in prelay geese, suggesting that these genes may be involved in laying function or lipid metabolism related to egg formation.

Genetic variation and phylogenetics of Lanyu and exotic pig breeds in Taiwan analyzed by nineteen microsatellite markers.

The Lanyu pig is an indigenous miniature pig breed on Lanyu Islet near Taiwan, with a mitochondrial DNA genetic lineage remote from Asian and European pig breeds. The unknown population genetic structure and increased inbreeding among the small population of conserved Lanyu pigs is now of great conservation concern. Additionally, the presence for more than a century of exotic pig breeds in Taiwan has made gene introgression from exotic pig breeds into Lanyu pigs very possible. The present study thus aimed to investigate nuclear genetic variation within the conserved Lanyu pigs and the phylogenetic relationship and possible genetic introgression between Lanyu and exotic pig breeds by determining the polymorphism of 19 microsatellite loci. In the neighbor-joining tree constructed from 7 pig breeds based on Cavalli-Sforza and Edward chord genetic distances, 3 major clades were recognized, in which the Asian and European breeds were separately clustered into 2 clades with a 93.0 and 99.9% bootstrap confidence value, respectively. All individuals of the Lanyu breed formed a unique subclade within the Asian clade based on the distance of the proportion of shared alleles, -ln(ps), suggesting that the Lanyu breed possesses a unique nuclear genetic structure and that no nuclear gene introgression from exotic breeds into the conserved Lanyu pigs has occurred in recent history. Fifteen of 19 microsatellite loci deviated from Hardy-Weinberg equilibrium (by Wright's statistic), suggesting a significant loss of heterozygosity in the conserved population. The valuable nuclear genetic structure and phylogenetic information should assist future conservation and population management of Lanyu pigs.

Interpopulation and intrapopulation maternal lineage genetics of the Lanyu pig (Sus scrofa) by analysis of mitochondrial cytochrome b and control region sequences.

The Lanyu pig is an indigenous breed from the Lanyu Islet, which is southeast of Taiwan. Two herds of Lanyu pigs were introduced from the Lanyu Islet into Taiwan in 1975 and 1980. The current population of conserved Lanyu pigs consists of only 44 animals with unknown genetic lineage. The Lanyu pig possesses a distinct maternal genetic lineage remote from Asian and European pigs. The present study aimed to understand the phylogenetic relationship among conserved Lanyu, Asian, and European type pigs based on the cytochrome b coding gene, to ascertain the maternal lineage and genetic diversity within the conserved Lanyu pigs, and to address whether genetic introgression from exotic or Formosan wild pigs had occurred in the conserved Lanyu pigs. Entire mitochondrial genomes of both types of Lanyu pig comprised 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes. Only 2 haplotypes of the mitochondrial DNA (mtDNA) control region and cytochrome b were identified in the conserved Lanyu pig herds. When maximum likelihood trees were constructed, the Type I Lanyu mitochondrial genes formed a unique clade with a large pairwise distance of both cytochrome b and the control region from Asian and European type breeds, Formosan wild pigs, and exotic breeds. Significant loss of genetic diversity of mtDNA within the conserved Lanyu pigs was demonstrated by low haplotype and nucleotide diversities, supported by Fu and Li's D* neutrality test (1.44055; P < 0.05). The mtDNA control region sequences of extant pigs in the Lanyu Islet, however, showed high haplotype and nucleotide diversity, and clustered with exotic pigs. These results indicate no maternal lineage mtD-NA gene introgression from Formosan wild pigs and introduced exotic pigs to conserved Type I Lanyu pigs, and a severe loss of heterozygosity of mtDNA in conserved Lanyu pigs. The remaining extant pigs on the Lanyu Islet have been introgressed with exotic breeds. Strategies for future conservation of native Lanyu pigs are now even more urgent and important.

The type I Lanyu pig has a maternal genetic lineage distinct from Asian and European pigs.

The Lanyu pig is an indigenous breed from Lanyu Islet, located south-east of Taiwan, with phenotypic characteristics distinctive from other pig breeds in Asia and Europe. Based on geographic considerations, the Lanyu pig may have originated from mainland China, Austronesia or the Ryukyu Islands. In the present study, polymorphism of the mitochondrial DNA control region sequence was used to clarify phylogenetic relationships among two herds of Lanyu pigs imported before 1980 from Lanyu Islet into Taiwan and reared in isolation on two different farms. Two distinct mitochondrial control region haplotypes were found. The type I Lanyu sequence appeared independently as a unique clade different from Asian and European pig sequences, while the type II Lanyu sequence was clustered within the major Asian clade. The pairwise distances between the major Asian clade vs. the type I Lanyu and European clades were 0.01726 +/- 0.00275 and 0.01975 +/- 0.00212 changes per site respectively. Estimates of divergence time suggest that the type I Lanyu sequence split from the major Asian pig clade in prehistoric times. The type II Lanyu mtDNA shares a close genetic lineage with Japanese Satsuma and New Zealand Kune Kune mtDNA with pairwise distances of 0.00095 +/- 0.00000 and 0.00192 +/- 0.00000 respectively, indicating gene flow between Lanyu Islet, Japan and Oceania in recent times. Together these results indicate that the type I Lanyu pig has a genetic lineage separate from Asian-type pigs, while the type II Lanyu sequence may represent a more recent introgression of modern Asian pigs.