Suppression of Grb2 expression improved hepatic steatosis, oxidative stress, and apoptosis induced by palmitic acid in vitro partly through insulin signaling alteration

In this study, we aimed to study the role of growth factor receptor-bound protein 2 (Grb2) in palmitic acid-induced steatosis and other “fatty liver” symptoms in vitro. HepG2 cells, with or without stably suppressed Grb2 expression, were incubated with palmitic acid for 24 h to induce typical clinical “fatty liver” features, including steatosis, impaired glucose metabolism, oxidative stress, and apoptosis. MTT and Oil Red O assays were applied to test cell viability and fat deposition, respectively. Glucose uptake assay was used to evaluate the glucose utilization of cells. Quantitative polymerase chain reaction and Western blot were used to measure expressional changes of key markers of insulin signaling, lipid/glucose metabolism, oxidative stress, and apoptosis. After 24-h palmitic acid induction, increased fat accumulation, reduced glucose uptake, impaired insulin signaling, enhanced oxidative stress, and increased apoptosis were observed in HepG2 cells. Suppression of Grb2 in HepG2 significantly reduced fat accumulation, improved glucose metabolism, ameliorated oxidative stress, and restored the activity of insulin receptor substrate-1/Akt and MEK/ERK pathways. In addition, Grb2 deficiency attenuated hepatic apoptosis shown by reduced activation of caspase-3 and fluorescent staining. Modulation of Bcl-2 and Bak1 also contributed to reduced apoptosis. In conclusion, suppression of Grb2 expression in HepG2 cells improved hepatic steatosis, glucose metabolism, oxidative stress, and apoptosis induced by palmitic acid incubation partly though modulating the insulin signaling pathway.     

High-level expression of a xylanase gene from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5?l fermentor culture, the total extracellular protein was 8.1?g?l^?1 with an activity of 52,940?U?ml^?1. The enzyme was purified to homogeneity with a recovery of 48?%. The recombinant xynA was optimally active at 75?°C, as measured over 10?min, and at pH 7. The enzyme was stable up to 80?°C for 30?min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).     

A novel homodimeric lectin from Astragalus mongholicus with antifungal activity

A novel lectin (AMML) was isolated from a Chinese herb, i.e., the roots of Astragalus mongholicus, using a combination of ammonium sulfate fraction and ion exchange chromatographies. The molecular mass of intact AMML was determined to be 66,396 Da by MALDI-TOF mass spectrometry and 61.8 kDa by gel filtration, respectively. AMML was a dimeric protein composed of two identical subunits each with a molecular mass of 29.6 kDa. The lectin was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified lectin hemagglutinated both rabbit and human erythrocytes, and showed preference for blood types O (native) and AB (trypsin-treated). Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives with pronounced preference for lactose (3.13 mM). N-terminal amino acid sequence of AMML was determined as ESGINLQGDATLANN. The optimal pH range for lectin activity was between pH 4.5 and 7.5, and the lectin was active up to 65 degrees C. It also exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.     

Comparison of Treatment Safety and Patient Survival in Elderly versus Nonelderly Patients with Advanced Hepatocellular Carcinoma Receiving Sorafenib Combined with Transarterial Chemoembolization: A Propensity Score Matching Study

AimsThis retrospective study was carried out to compare the outcomes between elderly (≥70 years of age) and nonelderly patients (<70 years of age) with advanced hepatocellular carcinoma (HCC) who received sorafenib combined with transarterial chemoembolization (TACE).Methods88 patients with a confirmed diagnosis of advanced HCC were enrolled in this study. Of these, 24 elderly patients were matched with 48 nonelderly patients at a 1:2 ratio using propensity score matching to minimize selection bias. The related adverse events and survival benefits were compared between the two groups.ResultsSorafenib combined with TACE was equally well tolerated in both age groups, and grade 3 or 4 adverse events were similarly observed in 54.2% of elderly and 50.0% of nonelderly patients (P = 0.739). There were no significant differences in survival time between the elderly and nonelderly patients (P = 0.876). Significant prognostic factors for overall survival as identified by multivariate analysis were the Child–Pugh score and portal vein invasion.ConclusionsSorafenib combined with TACE may be well tolerated and effective in elderly patients with advanced HCC. Age alone is not a parameter for the treatment of advanced HCC patients.     

Precise mapping of a quantitative trait locus interval for spike length and grain weight in bread wheat (Triticum aestivum L.)

The spike characteristics length, spikelet density and fertile floret number are related yield components and are important in cereal improvement. QSpl.nau-2D is a major quantitative trait locus controlling spike length (SPL) detected in the recombinant inbred line population developed by crossing wheat (Triticum aestivum) cultivars Nanda2419 with Wangshuibai. In this study, to validate its genetic effect and determine its precise location, QSpl.nau-2D’s near-isogenic line (NIL) was developed using Mianyang99-323 as the recurrent parent through marker-assisted selection. Field trials showed that the NIL not only had significantly longer spikes on average than the recurrent parent but also had significantly higher grain weight, but did not differ in spikelet number and kernel number per spike. In the F2 population derived from a cross of the NIL with Mianyang99-323, QSpl.nau-2D functioned like a single gene and conditioned the SPL in a partially dominant manner, and was thus designated as HL1 (for head length). To precisely map HL1, 89 recombinants, consisting of 11 genotypes, were identified in the NIL-derived F2 population of 674 plants by using markers in the Xwmc25–Xgpw4080 interval. Phenotyping these lines showed that the introduction of a 0.9-cM interval flanked by Xcfd53 and DG371 in Nanda2419 resulted in longer spikes and a higher grain weight in the NIL. The availability of markers closely linked to HL1 could facilitate its use in breeding programs.     

Sorafenib Combined with Transarterial Chemoembolization versus Transarterial Chemoembolization Alone for Advanced-Stage Hepatocellular Carcinoma: A Propensity Score Matching Study

Aims

The purpose of the present study was to compare the efficacies of transarterial chemoembolization (TACE) combined with sorafenib versus TACE monotherapy for treating patients with advanced hepatocellular carcinoma (HCC).

Methods

We enrolled 321 patients and selected 280 with advanced HCC (Barcelona Clinic Liver Cancer stage C) who underwent TACE therapy between February 2009 and February 2013. TACE alone (monotherapy group) was administered to 198 patients (70.7%), and the remaining 82 (29.3%) underwent repeat combined TACE and sorafenib therapy (combined group). To minimize selection bias, these latter 82 patients were matched using propensity-score matching at a 1∶2 ratio with 164 patients who received TACE monotherapy. The primary endpoints were overall survival (OS) and related subgroup analysis. The secondary endpoints were time to progression (TTP) and treatment-related adverse events.

Results

Of the respective patients in the combined and monotherapy groups, 64.6% and 49.2% had vascular invasion, 87.8% and 91.1% had extrahepatic metastasis, and 54.3% and 47.1% had both. In the propensity-score–matched cohort, the OS survival of the combined group was significantly higher compared with the monotherapy group (7.0 months vs. 4.9 months, respectively, P = 0.003). The TTP was significantly longer in the combined group (2.6 months vs. 1.9 months, respectively, P = 0.001). Subgroup analysis showed that the outcomes of patients with advanced HCC without main portal vein invasion who were treated with combined therapy were significantly better compared with those who received monotherapy (P<0.05). Univariate and subsequent multivariate analyses revealed that the addition of sorafenib was an independent predictor of favorable OS and TTP (adjusted hazard ratios, 0.63 and 0.62, respectively; P<0.05 for both).

Conclusion

Sorafenib plus TACE was more effective than TACE monotherapy for treating patients with advanced HCC without main portal vein invasion. Future trials with larger samples are required to validate these preliminary findings.