iTRAQ-based proteomic analysis of DMH-induced colorectal cancer in mice reveals the expressions of ß-catenin, decorin, septin-7, and S100A10 expression in 53 cases of human hereditary polyposis colorectal cancer.

PURPOSE: The aim of this study is to explore the roles of ß-catenin, decorin, septin-7, and S100A10 expression in colorectal cancer development. METHODS: Twenty-five BALB/c mice were divided into five groups; four groups were administrated N,N-dimethylhydrazine for 0, 10, 15, and 20 weeks, and one group was administrated normal saline for 20 weeks. The colons were collected for histopathological analysis. Protein samples prepared from the frozen colon tissues of mice treated with N,N-dimethylhydrazine for the different time points were evaluated using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled with the 2D liquid chromatography-tandem mass spectrometry analysis. Based on the proteomic analysis results, immunohistochemical staining of ß-catenin, decorin, septin-7, and S100A10 was performed in paraffin-embedded mice colorectal tissue, and 53 cases of human hereditary polyposis colorectal cancer samples. RESULTS: Colorectal cancer was observed in mice treated with N,N-dimethylhydrazine for 20 weeks, and adenomas were observed in mice subjected to the 10-, and 15-week treatments. Seventy-two differentially expressed proteins were involved in the development of cancer as per the iTRAQ and spectrometry analysis. In normal epithelium, adenoma, and cancer from human hereditary polyposis colorectal cancer, S100A10 expression (c2 = 100.989, P = 0.000) was highest in cancer, whereas decorin (c2 = 12.852, P = 0.002) and septin-7 (c2 = 66.519, P = 0.002) expressions were highest in the normal epithelium, which was confirmed via immunohistochemical staining. CONCLUSIONS: The subcellular localization of ß-catenin and decorin, septin-7, and S100A10 expressions are associated with the development of colorectal cancer in mice after N,N-dimethylhydrazine treatment and in human hereditary polyposis colorectal cancers.

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Agrobacterium-mediated transformation of the ß-subunit gene in 7S globulin protein in soybean using RNAi technology.

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

DNA repair gene XRCC3 variants are associated with susceptibility to glioma in a Chinese population.

The susceptibility to glioma is not well understood. It has been suggested that the X-ray cross complementing group 3 (XRCC3) gene influences the capacity to repair DNA damage, leading to increased glioma susceptibility. In this study, we evaluated the relationship between XRCC3 mutations and glioma risk. Genotypes were assessed in 389 Chinese glioma patients and 358 healthy controls. XRCC3 Thr241Met (rs861539) and 2 additional polymorphisms, rs3212112 (c.774+19T>G) and rs1799796 (c.562-14A>G), were directly sequenced. The frequency of the rs861539 T allele was significantly lower in the glioma group than in healthy controls [11.1 vs 17.7%, odds ratio = 0.62 (0.48-0.80), P < 0.001]; the frequencies of the CT or CT+TT genotypes differed between groups (18.5 vs 31%, 20.3 vs 33.2%, respectively). The frequency of the rs3212112 G allele was significantly higher in the glioma group than in healthy controls [15.8 vs 5.3%, odds ratio = 2.94 (2.07-4.17), P < 0.001]. The frequencies of the GT or TG+GG genotypes differed between groups (25.4 vs 7.8%, 28.5 vs 9.2%, respectively). This study demonstrates that the rs861539 and rs3212112 polymorphisms in the XRCC3 gene may influence the risk of glioma development in Chinese populations.

Analysis of quantitative trait loci for main plant traits in soybean.

Plant traits are important indices for regulating and controlling yield ability in soybean varieties. It is important to comprehensively study the quantitative trait locus (QTL) mapping for soybean plant traits, cloning related genes, and marker assistant breeding. In this study, 236 F2 generation plants and a derivative group were constructed by using Jiyu50 and Jinong18, obtained from Jilin Province. A total of 102 simple sequence repeat markers were used to construct a genetic linkage map. With 2 years of molecular and phenotypic data, QTL analyses and mapping were conducted for soybean maturity, plant height, main stem node, main stem branch, seed weight per plant, and more. Five main plant traits were analyzed via inclusive composite interval mapping using QTL IciMapping v2.2. Using one-dimensional scanning, a total of 30 QTLs were detected and distributed across 1 (A1), 4 (C2), and 12 (G). There were 9 linkage groups, including 16 major QTLs. Using two-dimensional scanning, 7 pairs of epistatic QTL interactions for maturity and plant height were detected in the soybean.

Identification of genes associated with the increased number of four-seed pods in soybean (Glycine max L.) using transcriptome analysis.

Seed number per pod is an important component of yield traits in soybean (Glycine max L.). In 2010, we identified a natural mutant with an increased number of four-seed pods from a soybean variety named 'Jinong 18' (JN18). Subsequent observations indicated that the trait was stably inherited. To identify and understand the function of genes associated with this mutant trait, we analyzed the genetic differences between the mutant (JN18MT01) and source variety (JN18) by transcriptome sequencing. Three types of tissues, axillary buds, unfertilized ovaries, and young pods at three different growth stages, V6, R1, and R3, were analyzed, respectively. The sequencing results yielded 55,582 expressed genes and 4183 differentially expressed genes (DEGs). Among these, the log2 ratio value of 162 DEGs was >10, and 13 DEGs had overlapping expression at three different growth stages. Comparisons of DEGs among three different growth stages yielded similar results in terms of the percentage of genes classified into each gene ontology (GO) category. DEGs were classified into 25 different functional groups in clusters of orthologous groups analysis. Proportions of the main functional genes differed significantly over developmental stages. A comparison of enriched pathways among the three developmental stages revealed that 646 unigenes were involved in 103 metabolic pathways. These results show that the development of four-seed pods is associated with a complex network involving multiple physiological and metabolic pathways. This study lays the foundation for further research on cloning and on the molecular regulation of genes related to the four-seed pod mutation.

Genetic analysis of the major gene plus polygene model in soybean resistance to Leguminivora glycinivorella.

In order to investigate the genetic characteristics of soybean Leguminivora glycinivorella resistance and to improve soybean resistance insectivorous breeding efficiency by applying the multi-generation joint analysis method of the major gene plus polygene model, 5 pedigrees and generations (P1, F1, P2, F2, and F2:3) were used as the materials to perform the soybean L. glycinivorella resistance multi-generation joint analysis. The results showed that soybean resistance to L. glycinivorella was controlled and inherited by an additive major gene plus additive, dominant polygene. The major gene had a negative additive effect (d = -0.1633). The combination of the anti-L. glycinivorella genes showed negative heterosis. Because the polygene additive effects were positive, the polygene effects would increase the insect herbivory rate in the F1 generation. This hybrid combination showed an insect herbivory rate polygenic heritability of 21.9556 and 54.3490% in the F2 and F2:3 pedigrees, which presented a high heritability. Therefore, it was appropriate to perform the selective breeding of the insect herbivory rate in the late generation.

Mutational analyses of the TSC1 and TSC2 genes in cases of tuberous sclerosis complex in Chinese Han children.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurogenetic disorder characterized by hamartomas in multiple organs and is caused by a wide spectrum of mutations in 1 of 2 causative genes (TSC1 or TSC2). Here, we present mutational analyses of the TSC1 and TSC2 genes in 4 cases of TSC in Chinese Han children, including 2 familial and 2 sporadic cases, using PCR and DNA sequencing of the entire coding region as well as exon-intron boundaries of these genes. Three mutations were identified in the TSC2 gene. Of these mutations, 2 mutations (c.3312-3313delGA and c.45delT) were novel, and the 3rd mutation (c.5238-5255del) was previously reported in Chinese Han and other populations. These mutations were not present in healthy family members or in 100 unrelated normal controls. The identification of these mutations in this study further expands the spectrum of known TSC2 gene mutations and contributes to prenatal molecular diagnosis and preimplantation genetic testing of TSC.

Molecular cloning and single nucleotide polymorphism analysis of IGF2a genes in the common carp (Cyprinus carpio).

We studied whether two IGF2 transcripts in common carp are similar to those found in zebrafish. The full-length IGF2a cDNA contains a 5'-terminal untranslated region (UTR) of 105 bp, a 3'-terminal UTR of 1358 bp and an open reading frame of 612 bp, which encodes a 206-amino acid protein. A 6614-bp full-length IGF2a DNA molecule, including the 5'-flanking region, was isolated. Genomic DNA structure analysis revealed that the IGF2a gene contains four exons and three introns. Bioinformatics analysis indicated that the proteins encoded by IGF2a genes in common carp have one signal peptide and one apparent transmembrane region. Bootstrapping was performed 1000 times to obtain support values for each branch. The common carp IGF2a were clustered in one group, while the outgroup (common carp IGF1) clustered in another group. We identified two new single nucleotide polymorphisms in intron 2 of the gene. One polymorphism, A/N, can be found only in the Huanghe carp. The other polymorphism, C/N, can be found in both male Huanghe carp × female Heilongjiang carp and male Huanghe carp × female Jian carp. The second polymorphism, C/N, is primarily transferred from the male and may be related to heterosis.

Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line.

HepG2.2.15 cell is a widely used cell model for studying HBV (hepatitis B virus) in vitro. In these cells, the HBV genome is integrated in several sites of HepG2 cellular DNA. These multiple copies may have some influence on the cellular processes. We constructed a new plasmid, pSEH-Flag-HBV, and transfected it into HepG2 cells, and then screened it with hygromycin. We then used ELISA, PCR, and RT-PCR to detect the expression of HBV in these cell lines. A cell line that stably expressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) was established. Using Southern blotting analysis, we found that the HBV genome was integrated as a single copy in the cellular DNA. This cell line will be a useful alternative model for HBV studies.

Leishmania mexicana: the circular DNA 1 (CD1) element contains genes encoding nucleotide-binding protein.

OBJECTIVE: To determine the nucleotide sequence of cloned CD1 fragments from Leishmania mexicana and find ORFs predicted to have protein coding function. METHODS: CD1 element was separated by CHEF and recovered by agarase, and the digested CD1 fragments were cloned into pZero vector. Nucleotide sequences were determined by the dideoxy chain termination method with the automatic sequencing system ALF using the M13 universal primers. Sequences were analyzed using GCG-PCGENE computer programs. RESULTS: The sequence with 4,385 nucleotides was determined and two ORFs were considered to have protein coding function (encoding nucleotide-binding protein). CONCLUSION: Genes encoding nucleotide-binding protein were identified from the amplified CD1 element of Leishmania mexicana.

Reciprocal relations for transmission coefficients: theory and application.

The authors present a rigorous proof of certain intuitively plausible reciprocal relations for time harmonic plane-wave transmission and reflection at the interface between a fluid and an anisotropic elastic solid. Precise forms of the reciprocity relations for the transmission coefficients and for the transmitted energy fluxes are derived, based on the reciprocity theorem of elastodynamics. It is shown that the reciprocity relations can be used in conjunction with measured values of peak amplitudes for transmission through a slab of the solid (water-solid-water) to obtain the water-solid coefficients. Experiments were performed for a slab of a unidirectional fiber-reinforced composite. Good agreement of the experimentally measured transmission coefficients with theoretical values was obtained.