Production of Camellia oleifera Abel Seed Oil for Injection: Extraction, Analysis, Deacidification, Decolorization, and Deodorization.

Camellia seed oil (CSO), as a nutrient-rich edible oil, is widely used in foods, cosmetics, and other fields. In this work, the extraction, deacidification, decolorization, and deodorization processes of CSO were respectively optimized for meeting injectable oil standards. The results showed that the CSO extraction rate reached the highest level of 94% at optimized conditions (ultrasonic time, 31.2 min; reaction pH, 9.2; and reaction time, 3.5 h). The physicochemical indexes of CSO and 10 other vegetable oils were evaluated by the principal component analysis method, and the overall scores of vegetable oils were ranked as camellia seed oil > olive oil > rice oil > peanut oil > sesame oil > corn oil > soybean oil > sunflower oil > rapeseed oil > walnut oil > flaxseed oil. The physicochemical indicators of CSO were the most ideal among the 11 vegetable oils, which means that CSO is suitable as an injectable oil. Through the optimized processes of the deacidification, decolorization, and deodorization, the CSO acid value was reduced to 0.0515 mg KOH/g, the decolorization rate reached a maximum of 93.86%, and the OD430 was 0.015, meeting the requirement (≤0.045 of OD430) of injectable oil. After the deodorization process, these parameters of the refractive index, acid value, saponification value, iodine value, absorbance, unsaponifiable, moisture and volatiles, fatty acid composition, and heavy metal limits all met the pharmacopoeia standards of injectable oil in many countries and regions. The possibility of CSO as an injectable oil was first verified through refining-process optimization and nutritional index analysis, providing an important technical reference for the high-value utilization of vegetable oil.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM) is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, an SAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

[Effect of SARI Overexpression on Apoptosis of CBFL Cells and Its Mechanism].

OBJECTIVE: To investigate the effect of SARI overexpression on the proliferation and apoptosis of core binding factor leukemia (CBFL) cells and explore the potential molecular mechanisms. METHODS: C-KIT N822K mutation status in Kasumi-1 cell line was detected by exon 17 sequencing. Then the SARI lentiviral vector (pGC-FU-SARI) was constructed, meanwhile Kasumi-1 cells were transfected with the SARI lentiviral vector. Quantitative PCR and Western blot were employed to identify efficacy of SARI overexpression after the transfection of cells. Cells were divided into three groups, including the cells infected with pGC-FU-SARI (OE group), the cells infected with pGC-FU-GFP (NC group) and the untreated cells (blank control group). Cell proliferative activity was tested by MTT assay, cell apoptosis was measured by flow cytometry (FCM) and the expression of apoptosis-related proteins: BCL-2,BAX,Cyto C,Caspase 9,Caspase 3,cleaved-Caspase 3,PARP and cleaved-PARP as well as PI3K/Akt pathway proteins: PI3K（p85）,p-PI3K（p85）,Akt and p-Akt were detected by Western blot. RESULTS: The Kasumi-1 cells were detected to bear c-KIT N822K (T>A) mutation. The Kasumi-1 cells with SARI was overexpression were construeted successfully. Compared with NC group, the cell proliferation was decreased and cell apoptosis was increased; BCL-2 expression was reduced, BAX expression was enharued; cyto C expression appeared; the expression of Caspase 9 and Caspase 3 was down-regulated, the expression of cleaved Caspase 3 was up-regulated; the PARP expression was decreased, cleaved PARP expression was increased; the phosphorylation level of PI3K/Akt pathway proteins: p-PI3K/PI3K, p-Akt/Akt was down-regulated in OE group (P<0.05). CONCLUSION: SARI gene may suppress the proliferation of CBFL cells, and induce their apoptosis through the mitochondrial pathway, which may be related with the inhibition of PI3K/Akt pathway.

Construction of industrial baker's yeast with high level of cAMP.

Cyclic adenosine monophosphate (cAMP) plays an important role in modulating the activity of microbe cell. In this study, PKA (protein kinase A) activity was weakened through truncation of TPK2 promoter (-150 bp and -300 bp) and gene deletion of BCY1 (encodes the regulatory subunit of PKA), TPK1 and TPK3, generating strains BY9a-T2-150 and BY9a-T2-300, respectively. High-performance liquid chromatography analysis showed cAMP levels in BY9a-T2-150 and BY9a-T2-300 were increased by 5- and 18-fold, respectively, compared with that of parent strain, BY9a. The expression levels of TPK2 gene in two engineered strains were decreased by 95% and 97% compared with that of BY9a, respectively. The PKA activity reflected by heat resistance of engineered strains enhanced compared with parent strain BY9a. This study show a new method to increase the intracellular cAMP concentration in industrial yeast by fine-tuning of PKA activity, without influence in growth and fermentation properties. PRACTICAL APPLICATIONS: cAMP as the "second messenger," is essential for plant, animal, and microorganisms and human life. But its synthesis is still limited by expensive cost and time-consuming method. We constructed the industrial baker's yeast with high level of cAMP and desired to be used to produce functional food for relaxing smooth muscle, expanding blood vessels, improving liver function, and promoting nerve regeneration and as a food additive for treating hyperthyreosis and hepatopathy. The methods of two step homologous recombination and backcross operated in this study eliminate the exogenous gene in engineered strains, made it safety to be used in food production. Fine-tuning of PKA activity in engineered strains ensure produce high level of cAMP and exhibit normal growth performance in engineering strains. Therefore, this work is significant in functional foods product and has the potential to be used in practical application.

Inhibition of N822K T>A mutation-induced constitutive c-KIT activation in AML cells triggers apoptotic and autophagic pathways leading to death.

Background: The D816V mutation of c-KIT can constitutively activate tyrosine kinase, thereby promote core binding factor acute myeloid leukemia (CBF-AML) cell proliferation and inhibit apoptosis. Previous studies have indicated similar proliferation and apoptosis between N822K and D816V mutations.The current study aims to determine the occurrence and potential functions of N822K mutation-induced c-KIT activation in AML cells, and explore possible mechanisms of poor prognosis of CBF-AML. Methods: c-KIT N822K mutation status in AML cells was determined by exon 17 sequencing. The level of c-KIT expression was detected by flow cytometry (FCM) and colony formation was assessed after hu-SCF stimulation. After exposure to sunitinib (a kind of tyrosine kinase inhibitor, TKI), cell proliferation inhibition was tested by MTT, cell cycle and apoptosis were measured by FCM, autophagy was assessed by fluorescence microscopy and immunoblotting. Results: Kasumi-1 cell line was detected to bear c-KIT N822K (T>A) mutation. After hu-SCF stimulation, CD117 expression was decreased and the colony formation efficiency was not altered in Kasumi-1 cells. After sunitinib inhibited the c-KIT activity, the colony formation efficiency was reduced, and the half-maximal inhibitory concentration (IC50) of sunitinib was low (0.44±0.17µM) at 48 hours. Moreover, cells were arrested in G0/G1 phase, corresponding to an increase of apoptosis ratio. Acidic vesicular organelles (AVO) were observed along with an altered expression of autophagy-related proteins in Kasumi-1 cells. Conclusions: Our data indicated that inhibition of N822K T>A mutation-induced constitutive c-KIT activation in AML cells triggered apoptotic and autophagic pathways leading to death, and c-KIT N822K mutation may have clinical application as a CBF-AML treatment target.

Increase ethyl acetate production in Saccharomyces cerevisiae by genetic engineering of ethyl acetate metabolic pathway.

Ethyl acetate has attracted much attention as an important chemical raw material and a flavor component of alcoholic beverages. In this study, the biosynthetic pathway for the production of ethyl acetate in Chinese liquor yeast was unblocked. In addition to engineering Saccharomyces cerevisiae to increased intracellular CoA and acetyl-CoA levels, we also increased the combining efficiency of acetyl-CoA to ethanol. The genes encoding phosphopantothenate-cysteine ligase, acetyl-CoA synthetase, and alcohol acetyltransferase were overexpressed by inserting the strong promoter PGK1p and the terminator PGK1t, respectively, and then combine them. Our results finally showed that the ethyl acetate levels of all engineering strains were improved. The final engineering strain CLy12a-ATF1-ACS2-CAB2 had a significant increase in ethyl acetate yield, reaching 610.26 (± 14.28) mg/L, and the yield of higher alcohols was significantly decreased. It is proved that the modification of ethyl acetate metabolic pathway is extremely important for the production of ethyl acetate from Saccharomyces cerevisiae.

Comprehensive comparison of three different immunosuppressive regimens for liver transplant patients with hepatocellular carcinoma: steroid-free immunosuppression, induction immunosuppression and standard immunosuppression.

The different choices of immunosuppression (IS) regimens influenced the outcomes of liver transplantation. Steroid was applied as a standard IS to prevent and treat rejections. However, steroid-related complications were increasingly prominent. This study compared the efficacy and safety of standard IS regimens with the efficacy and safety of steroid-free IS regimen and induction IS regimen in Chinese liver transplantation recipients for hepatocellular carcinoma (HCC). A total of 329 patients who underwent liver transplantation from January 2008 to December 2012 were retrospectively reviewed. Three different groups of patients received standard triple-drug IS regimen of steroid, tacrolimus (TAC) and mycophenolate mofetil (MMF) (triple-drug regimen group; n=57), induction-contained IS regimen of basiliximab, steroid, TAC and MMF (BS group; n=241), and induction-contained and steroid-free regimen of basiliximab, TAC and MMF (SF group; n=31), respectively. There were no significant differences in terms of patient, tumor-free and graft survival rates. The acute rejection rate and rejection time were equivalent in different groups. But compared with BS group, higher incidences of biliary complications (11.52% vs. 30.77%, p=0.013) and graft dysfunction (0.48% vs. 13.64%, p=0.003) were observed in SF group. Furthermore, compared with the two groups, incidence of pleural effusion was also higher in SF group (15.79%, 11.96% vs. 45.45%, respectively, both p<0.01). And a trend towards less proportion of De novo diabetes was revealed in SF group. Although it was found that patient, tumor-free and graft survival rates were equivalent among three IS regimens, higher incidences of complications were demonstrated in steroid-free regimen in patients for HCC. These findings suggested that steroid-free IS regimen has no clear advantages in comparison with standard IS regimens for liver transplant recipients with HCC and the postoperative complications should be treated with concentrated attention.

The effect of ABCB1 C3435T polymorphism on pharmacokinetics of tacrolimus in liver transplantation: a meta-analysis.

OBJECTIVES: The effect of ABCB1 C3435T SNP on the pharmacokinetics of immunosuppressive drug tacrolimus in different studies was conflicting. So a meta-analysis was employed to study the correlation of ABCB1 C3435T SNP and the pharmacokinetics of tacrolimus at different post-transplantation times. METHOD: Several studies about ABCB1 C3435T polymorphism and the pharmacokinetics of tacrolimus were collected through the search on PubMed and the Cochrane Library. After the extraction of pharmacokinetic parameters from these studies, a meta-analysis was performed on the software STATA version 11.0. RESULTS: A total of 9 studies were adopted including 558 liver transplant recipients. For the dose of tacrolimus, the subjects with wild-type CC had a significantly higher tacrolimus dose than homozygous mutated genotype TT within 1 week (WMD=0.01 (0.00, 0.02), P=0.014) and the similar result in recipients with heterozygous CT compared with TT after transplantation for 1 month (WMD=0.01 (0.00, 0.02), P=0.002). For the tacrolimus concentration/dose ratio, subjects with CT had higher C/D ratio than those with CC and TT at different post-transplantation times. A subgroup analysis based on different ethnic populations was also carried out. Donors' genotypes were also considered in this meta-analysis. CONCLUSION: Through this meta-analysis for the including studies about the pharmacokinetics of tacrolimus and ABCB1 C3435T SNP, several significant associations were obtained. Particularly, the Caucasians showed more significant associations between the C/D ratio and ABCB1 C3435T polymorphism; however, the correlations were not steady at different post-transplantation times.

Electrospinning-based synthesis of highly ordered mesoporous silica fiber for lab-in-syringe enrichment of plasma peptides.

Silica fiber with highly ordered mesoporous structure and continuously long fibrous property was synthesized on a large-scale for the first time. It can be applied to the rapid (less than 3 min) and effective enrichment of endogenous peptides with a novel lab-in-syringe approach.