Low CPEB1 levels may predict the benefit of 5-fluorouracil treatment in patients with colon or stomach adenocarcinoma.

Background: For patients with colon or stomach adenocarcinoma, 5-fluorouracil (5-FU) is an essential component of systemic chemotherapy in the palliative and adjuvant settings. The post-transcriptional regulatory factor cytoplasmic polyadenylation element-binding protein 1 (CPEB1) has been reported to be linked to tumor metastasis. This study aimed to investigate the relationship between CPEB1 expression and 5-FU treatment response in patients with colon and stomach adenocarcinomas. Methods: The expression of CPEB1 in stomach adenocarcinoma and colorectal cancer (CRC) tissues and in cell lines was determined by quantitative real-time PCR (qRT-PCR) and immunohistochemistry analyses. Transwell assays were employed to analyze the effects of CPEB1 on the migration and invasion abilities of gastric cancer (GC) and CRC cells. Results: The expression levels of CPEB1 were increased in colon and stomach adenocarcinoma and were negatively correlated with malignancy and poor patient survival. Data suggested that patients with CRC or GC who had strong CPEB1 expression responded poorly to 5-FU treatment. Furthermore, knockdown of CPEB1 inhibited the migration and invasion of CRC and GC cells via a mechanism involving decreased expression of matrix metalloprotein (MMP)2, 7, and 9. Finally, our methylated RNA immunoprecipitation PCR (meRIP qPCR) data suggested that the increased CPEB1 expression in colon and stomach adenocarcinomas might be mediated by FTO (FTO alpha-ketoglutarate dependent dioxygenase)-dependent m6A demethylation of CPEB1 mRNA. Conclusions: Our results indicate that the level of CPEB1 expression may be valuable for predicting the benefit of 5-FU treatment for patients with colon and stomach adenocarcinomas. We therefore propose that low CPEB1 expression may represent a novel biomarker for personalized 5-FU therapy.

Vaccination influences the evolution of classical swine fever virus.

Classical swine fever is a serious, economically damaging disease caused by classical swine fever virus (CSFV). The CSFV is composed of two clades, according to phylogenetic estimates. Attenuated live vaccine such as HCLV, has been widely used to protect pigs from CSFV, but the influence of vaccination on the evolution of CSFV has not been studied. We conducted a systemic analysis of the impact of vaccination on the evolution of CSFV by comparing vaccine-related and non-vaccine-related CSFV groups. We found that vaccination may affect strain diversity and immune escape through recombination and point mutation. We also found that vaccination may influence the population dynamics, evolutionary rate and adaptive evolution of classical swine fever virus. Our evidence suggests that the vaccination might also change host adaptation through influencing codon usage of the virus in swine. These findings suggest that it is necessary to avoid excessive use of CSFV attenuated vaccines.

Identification of two severe fever with thrombocytopenia syndrome virus strains originating from reassortment.

Recently, a novel bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), was isolated in central China. The virus can cause multi-clinical symptoms: severe fever, thrombocytopenia, leukocytopenia, with a mortality rate of ~10%. Several studies show that SFTSV could undergo rapid evolution via gene mutation and homologous recombination. However, as an important evolutionary force for segmented-genome viruses, reassortment has not been reported in SFTSV. In this study, we identified two SFTSV strains of which the S segment has different origin from M and L, suggesting that reassortment might be potential force driving rapid change of SFTSV. This result might shed new light on the evolutionary behavior of the novel virus.

Genetically switched D-lactate production in Escherichia coli.

During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative D-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created. When the genetic switch was turned off at 33°C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42°C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33°C with a short thermo-induction at 42°C and then switched to the production phase at 42°C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8 g/l D-lactate with an increased oxygen-limited productivity of 0.89 g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.

Fine tuning the transcription of ldhA for D-lactate production.

Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli). Twelve ldhA genes with sequentially shortened chromosomal upstream regions were cloned in an ldhA deletion, E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA). The varied ldhA upstream regions were further analyzed using program NNPP2.2 (Neural Network Promoter Prediction 2.2) to predict the possible promoter regions. Two-phase fermentations (aerobic growth and oxygen-limited production) of these strains showed that shortening the ldhA upstream sequence from 291 to 106 bp successively reduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase. Simultaneously, oxygen-limited lactate productivity was increased during the second phase. The putative promoter downstream of the -96 site of ldhA could function as a transcriptional promoter or regulator. B0013-080C/pTH-rrnB-ldhA8, with the 72-bp upstream segment of ldhA, could be grown at a high rate and achieve a high oxygen-limited lactate productivity of 1.09 g g(-1) h(-1). No transcriptional promoting region was apparent downstream of the -61 site of ldhA. We identified the latent transcription regions in the ldhA upstream sequence, which will help to understand regulation of ldhA expression.

Improvement of D-lactate productivity in recombinant Escherichia coli by coupling production with growth.

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O(2)-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O(2)-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.

Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli.

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.

[Changes of encephalic contents of neuropeptides in rats with vascular dementia].

OBJECTIVE: To investigate the changes of encephalic contents of somatostatin (SS) and arginine vasopressin (AVP) in rats with vascular dementia. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into three groups: vascular dementia group (VDMG), sham operation group (SOG) and control group (CG). The vascular dementia model was established by permanent bilateral vertebral artery occlusion through electric coagulation and shut-off the bilateral carotid arteries. The remember behavior of animals was tested and the contents of SS and AVP in various encephalic region (frontal cortex, temporal lobe, hippocampus, cerebral ganglion and corpora striatum) were determined with radioimmunoassay. RESULT: During the 15-day-long remembering test, the frequency of making mistakes in the VDMG was higher remarkably than that in SOG and CG (P<0.05); and the relative contents of SS were decreased in frontal area cortex, temporal lobe, hippocampus, cerebral ganglion and corpora striatum (P<0.01), while decrease of AVP contents was only detected in temporal lobe and corpora striatum (P<0.05). CONCLUSION: The disturbance of learning and memory function might be associated with SS and AVP after multiple cerebral infarction in the animals.

Cellular transcription modulator SMARCE1 binds to HBV core promoter containing naturally occurring deletions and represses viral replication.

Suppression of hepatitis B virus (HBV) replication, a causative agent for chronic hepatitis, is an effective approach to controlling disease progression. Host factors have a significant effect on viral replication efficiency and need to be better characterized. We have reported association between clinical virus load and deletions in HBV viral promoter. We showed here that HBV genome with such deletions led to decreased replication compared with wild type virus. Consistently, the promoter with deletion showed lower activity. A cellular transcription regulator recognizing the promoter with deletion was revealed in gel shift assay and subsequently identified as SMARCE 1 through DNA-protein array assay. The ability of SMARCE 1 in modulating the replication efficiency of HBV was further demonstrated. Taken together, our studies show a direct dependence of HBV on a host factor to modulate its replication efficiency, and provided a new platform for molecular characterization of mechanisms of disease outcome as a result of binding of new transcription factors to rearranged promoter sequences.

[Cloning of the gene encoding a thermostable alpha-amylase from bacillus licheniformis CICIM B0204 and functional identification of its promoter].

Thermostable alpha-amylase, catalyzing the hydrolyzation of starch to dextrin, maltose and glucose at higher temperature, is one of the most industrial important enzymes. Several species of Bacillus have been found and genetic improved to produce the thermostable alpha-amylases. In present study, a gene, amyL, coding for a thermostable alpha-amylase with its flanking sequences was cloned from an industrial Bacillus licheniformis CICIM B0204 by using a combination of routine polymerase chain reactions (PCR) and inverse PCR with a pair of initial primers derived from the highly conserved region of bacterial alpha-amylase genes and the functional identifications of the cloned amyL and the activities of its promoter and signal peptide in Escherichia coli were investigated. The amyL was composed of 1539 bp with 180 bp at upstream for its promoter and 160 bp at downstream for its terminator. The deduced mature peptide of the a-amylase was composed of 512 amino acid residues and its signal peptide 29 amino acid residues at N-terminal. The nucleotide and deduced amino acid sequences of amyL were extremely similarity to those from Bacillus species with three amino acid residues difference (Arg163-->Leu, Ser339-->Gly, Ala349-->Ser) comparison to that from a laboratory strain B. licheniformis 584. Under the control of T7 promoter, the structural region of amyL was functionally expressed in Escherichia coli. Additionally, the structural region of the gene coding for a beta-mannosidase from B. licheniformis CICIM B2004 was inframely inserted into the downstream of the promoter and signal sequence of amyL and expressed in E. coli. The amyL promoter and signal sequence was functionally directed the expression and secretion of the beta-mannosidase in E. coli cells with the expression level of 295 U/mL.