Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments.

Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8-9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change.

Analysis of tandem repeat units of the promoter of capsanthin/capsorubin synthase (Ccs) gene in pepper fruit.

Capsanthin/capsorubin synthase (Ccs) gene is a key gene that regulates the synthesis of capsanthin and the development of red coloration in pepper fruits. There are three tandem repeat units in the promoter region of Ccs, but the potential effects of the number of repetitive units on the transcriptional regulation of Ccs has been unclear. In the present study, expression vectors carrying different numbers of repeat units of the Ccs promoter were constructed, and the transient expression of the ß-glucuronidase (GUS) gene was used to detect differences in expression levels associated with the promoter fragments. These repeat fragments and the plant expression vector PBI121 containing the 35s CaMV promoter were ligated to form recombinant vectors that were transfected into Agrobacterium tumefaciens GV3101. A fluorescence spectrophotometer was used to analyze the expression associated with the various repeat units. It was concluded that the constructs containing at least one repeat were associated with GUS expression, though they did not differ from one another. This repeating unit likely plays a role in transcription and regulation of Ccs expression.

Effects of silencing key genes in the capsanthin biosynthetic pathway on fruit color of detached pepper fruits.

BACKGROUND: There are many varieties of carotenoids in pepper fruits. Capsanthin is a red carotenoid that gives mature pepper fruits their red color. The red color in pepper fruits is regulated mainly by the genes capsanthin/capsorubin synthase(Ccs), phytoene synthase(Psy), lycopene-ß-cyclase(Lcyb) and ß-carotene hydroxylase(Crtz). There has been very limited research work related to the development and change in the red color during fruit formation and when a certain gene or several genes are deleted. In this paper, we constructed viral vectors, using the tobacco rattle virus (TRV), to carry the target gene to infect detached pepper fruits, and observed the fruits' color change. We used real-time quantitative PCR to analyze the gene silencing efficiency. At the same time, HPLC was used to determine the content of capsanthin and carotenoids that are associated with capsanthin synthesis when key genes in the pepper fruits were silenced. RESULTS: These genes (Ccs, Psy, Lcyb and Crtz) were individually silenced through virus induced gene silencing (VIGS) technology, and pepper fruits from red fruit cultivars showed an orange or yellow color. When several genes were silenced simultaneously, the fruit also did not show the normal red color. Gene expression analysis by real-time quantitative PCR showed 70-80% efficiency of target gene silencing when using the VIGS method. HPLC analysis showed that the contents of carotenoids associated with capsanthin synthesis (e.g. ß-carotene, ß-cryptoxanthin or zeaxanthin) were decreased in varying degrees when silencing a gene or several genes together, however, the content of capsanthin reduced significantly. The synthesis of capsanthin was influenced either directly or indirectly when any key gene was silenced. The influence of the target genes on color changes in pepper fruits was confirmed via the targeted silencing of them. CONCLUSIONS: VIGS was a good method to study the molecular mechanism of pepper fruit color formation. By using virus induced gene silencing technology, capsanthin synthesis genes in pepper fruits were silenced individually or simultaneously, and pepper fruit color changes were observed. This provides a platform to further explore the molecular mechanism of pepper fruit color formation.

Effects of Chlorella extracts on growth of Capsicum annuum L. seedlings.

The long-term application of chemical fertilizers has caused to the farmland soil compaction, water pollution, and reduced the quality of vegetable to some extent. So, its become a trend in agriculture to find new bio-fertilizers. Chlorella extract is rich in amino acids, peptides, nucleic acids, growth hormones, potassium, calcium, magnesium, iron, zinc ions, vitamin E, B1, B2, C, B6, folic acid, free biotin and chlorophyll. Chlorella extract can promote biological growth, mainly by stimulating the speed of cell division, thereby accelerating the proliferation rate of cells and playing a role in promoting plant growth. Whether Chlorella extract can be used to improve the growth of pepper (Capsicum annuum), needs to be verified. In current study, a pepper variety 'Chao Tian Jiao' was used as experiment material, by determining the changes of the related characteristics after spraying the seedlings with Chlorella extract, and its effect on growth of Capsicum annuum plants was investigated. The results showed that the Chlorella extract significantly increased plant height of pepper seedlings (treatment: 32.2 ± 0.3 cm; control: 24.2 ± 0.2 cm), stem diameter (treatment: 0.57 ± 0.02 cm; control: 0.41 ± 0.03 cm) and leaf area (treatment: 189.6 ± 3.2 cm2; control: 145.8 ± 2.5 cm2). Particularly, the pepper seedlings treated with Chlorella extract, developed the root system in better way, significantly increased the chlorophyll a, and the activities of SOD, POD and CAT enzymes were also improved significantly. Based on our results, we can speculate that it is possible to improve the growth of Capsicum annuum seedlings and reduce the application of chemical fertilizers in pepper production by using Chlorella extract.

Transarterial chemoembolization combined with metformin improves the prognosis of hepatocellular carcinoma patients with type 2 diabetes.

Objective: The study aims to investigate the effect of metformin on Hepatocellular carcinoma (HCC) patients with type 2 diabetes mellitus (T2DM) who received transarterial chemoembolization (TACE) for the first time. Methods: From January 2016 to December 2019, T2DM patients diagnosed with HCC in Shandong Cancer Hospital and treated with TACE were included in this retrospective study. Overall survival (OS) and Progression-free survival (PFS) were compared between patients treated with metformin and other antidiabetics. Univariate and multivariate Cox regression models were used to evaluate the independent risk factors associated with OS and PFS. And sub-analysis was performed to investigate whether metformin could give a survival advantage in each Barcelona Clinic Liver Cancer (BCLC) stage of HCC. Propensity score matched (PSM) analyses based on patient and tumor characteristics were also conducted. Results: A total of 123 HCC patients with T2DM underwent TACE, of which 50 (40.65%) received treatment with metformin. For the whole cohort, the median OS (42 vs 32 months, p=0.054) and PFS (12 vs 7 months, P=0.0016) were longer in the metformin group than that in the non-metformin group. Multi-analysis revealed that BCLC stage, BMI (Body Mass Index), and metformin use were independent predictors of OS. Metformin use was independently associated with recurrence. After PSM, 39 matched pairs were identified. The use of metformin was associated with a numerically longer m OS (43 vs 35 months, P=0.183) than the use of other anti-diabetics. And the difference in median PFS (13 vs 7 months, p=0.018) between the metformin group and non-metformin group remained significant. Conclusion: The combination of transarterial chemoembolization and metformin may be associated with better OS and PFS in HCC patients with T2DM.

The role of bile acid subtypes in the diagnosis of cholangiocarcinoma.

OBJECTIVES: To find the potential biomarkers of cholangiocarcinoma, form a biomarker package, evaluate its efficiency, and validate it. METHODS: R software was used to analyze the differential expression of mRNAs between cholangiocarcinoma and adjacent nontumorous tissues, obtained from The Cancer Genome Atlas (TCGA), and enrich the KEGG pathway. Metabo-Profile Inc. performed the comprehensive bile acid profiling and quantitation. The training set concluded 20 cholangiocarcinoma and 20 nontumorous volunteers. Receiver operating characteristic (ROC) curve and accompanying area under the curve (AUC) was calculated. The top four bile acids formed a new biomarker package. The validation set included 15 cholangiocarcinoma and 15 nontumorous, and the sensitivity and specificity of the new biomarker package were tested. RESULTS: Gene expression of 36 cholangiocarcinoma and nine adjacent nontumorous tissues was obtained in January 2020. Totally 9887 differential genes were eligible (logFC ≥ 1 or ≤ -1, P < 0.05, and adjust P < 0.01). GO analysis showed that 20 KEGG pathways were enriched, including primary bile acid biosynthesis and bile secretion. Comprehensive bile acid profiling and quantitation showed 15 differential bile acid types, and the ROC-AUC was between 0.953 and 0.750. HDCA, isoLCA, bCDCA, and DCA were selected to form a biomarker package. The Logit (p = cholangiocarcinoma) = 7.898 - 3.70*(1isoLCA) - 0.444*(bCDCA) + 0.415*(HDCA) + 0.041*DCA. Its ROC-AUC was 0.944. In the validation set, the sensitivity was 0.933 and the specificity was 0.867. CONCLUSION: Bile acid types package was efficient to distinguish nontumorous population and cholangiocarcinoma. The difference might be associated to the downregulation of primary bile acid biosynthesis and bile secretion pathway of cholangiocarcinoma.

Modified percutaneous transhepatic papillary balloon dilation for patients with refractory hepatolithiasis.

BACKGROUND: Some patients with hepatolithiasis cannot tolerate surgery due to severe cardiac or pulmonary comorbidities, or cannot be endoscopically treated because of altered gastrointestinal anatomies. AIM: To propose a modified percutaneous transhepatic papillary balloon dilation procedure, and evaluate the clinical efficacy and safety of this modality. METHODS: Data from 21 consecutive patients who underwent modified percutaneous transhepatic papillary balloon dilation with hepatolithiasis were retrospectively analyzed. Using auxiliary devices, intrahepatic bile duct stones were pushed into the common bile duct and expelled into the duodenum with an inflated balloon catheter. The outcomes recorded included success rate, procedure time, hospital stay, causes of failure, and procedure-related complications. Patients with possible long-term complications were followed up for 2 years. RESULTS: Intrahepatic bile duct stones were successfully removed in 20 (95.23%) patients. Mean procedure time was 65.8 ± 5.3 min. Mean hospital stay was 10.7 ± 1.5 d. No pancreatitis, gastrointestinal, or biliary duct perforation was observed. All patients were followed up for 2 years, and there was no evidence of reflux cholangitis or calculi recurrence. CONCLUSION: Modified percutaneous transhepatic papillary balloon dilation was feasible and safe with a small number of patients with hepatolithiasis, and may be a treatment option in patients with severe comorbidities or in patients in whom endoscopic procedure was not successful.

Cloning and characterization of the CarbcL gene related to chlorophyll in pepper (Capsicum annuum L.) under fruit shade stress.

Light is an important environmental factor for fruit development and ripening in pepper plant. Fruit bagging is a significant agrotechnology practiced for the illumination regulation of fruits; some previous researches have shown that fruit bagging could improve the appearance and external quality of fruits and cause them to mature early. However, it would decrease the intrinsic qualities of fruits; especially, fruit bagging could decrease the content of capsanthin in peppers. On the basis of these details, fruit bagging was used as the method of fruit shade stress in this study to explore the characteristics and molecular mechanisms of pepper fruit's color change under shade stress. By using cDNA-AFLP under fruit shading, a fragment related to fruit color was obtained. Next, the full-length coding sequence of the gene was cloned from the pepper fruits. Homologous gene alignment confirmed that the gene has high homology with the rbcL gene, named CarbcL. The function of the CarbcL gene was identified through virus-induced gene silencing (VIGS); it was found that the fruit color changed completely from green to red except for some residue of green fleck when CarbcL gene was silenced, and the green color of fruits had not fully faded in the control group and the empty vector group. The combine determination of chlorophyll content showed that CarbcL was involved in the metabolic control of chlorophyll in pepper fruits; subsequently, HPLC was used to determine the content of capsanthin in pepper fruit which the CarbcL gene was silencing, and it was also found that the content of capsanthin decreased appreciably. These results further confirmed that CarbcL gene was involved in the adjustment of chlorophyll and capsanthin.