Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

Deep-sea water inhibits metastatic potential in HT-29 human colorectal adenocarcinomas via MAPK/NF-κB signaling pathway

A previous investigation showed that deep-sea water (DSW) can affect the expression of genes that regulate metastasis, including cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), urokinase plasminogen activator (uPA) and uPA receptor (uPAR), in HT-29 human colorectal adenocarcinomas. In the present study, we investigated the effects of DSW on inducible nitric oxide synthase (iNOS) expression and cell migration and also explored the mechanism of DSW-induced anti-metastatic potential in HT-29 human colorectal adenocarcinomas. Cytokine-induced expression of iNOS, which is highly expressed in colon cancer and enhances cancer growth and metastasis, was decreased in a hardness-dependent manner by DSW. Also, the wound healing assay revealed that DSW inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration in a hardness-dependent manner. DSW also decreased the phosphorylation of various MAPKs, including p38, ERK and JNK, and suppressed the nuclear translocation of NF-κB but not c-Jun. The results suggest that DSW may inhibit cancer cell growth related to iNOS overexpression and PKC-mediated cell migration in HT-29 human colorectal adenocarcinomas and the antimetastatic potential of DSW may be regulated by prevention of NF-κB nuclear translocation via inhibition of p38, ERK and JNK phosphorylation. In conclusion, the present investigation demonstrates that DSW inhibits cancer growth and metastasis via down-regulation of iNOS expression and the MAPK/NF-κB signaling pathway.     

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure

Cry2Ab, a pore‐forming toxin derived from Bacillus thuringiensis, is widely used as a bio‐insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250‐kDa prepore oligomer. Site‐directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.     

Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases

Hepatocellular carcinoma (HCC) is a very aggressive neoplasia requiring early and accurate diagnosis to improve patient outcomes with timely treatment. The liver is also very frequently colonized by metastases, and the most frequent differential diagnosis is HCC against intrahepatic cholangiocarcinoma or metastatic adenocarcinoma. Metabolomics is a powerful tool for identification of altered biomarkers in cancer, and to evaluate the efficacy of drug treatments. Here we analyzed by HILIC-MS/MS methylated arginines, basic amino acids (Arg, Cit, Orn), and their ratios in the extracts of primary HCC tissues, liver metastases from colorectal carcinoma (MET), cirrhotic related hepatitis-C-virus (CIR), and non-cirrhotic normal liver (NT) adjacent tissues. We found high levels of Arg (p < 0.0001) and Arg/Orn (p < 0.01) in MET compared to other tissues. In MET, compared to NT and CIR, Arg concentration was fivefold higher, while in HCC it was twofold higher. ADMA increased twofold compared to NT and CIR, while in HCC it was 50 % higher. Arg/Cit and ADMA/SDMA ratios were significantly higher in MET compared to NT and CIR (p < 0.005). Arg/Orn, Arg/Cit, and ADMA/SDMA ratios increased progressively from NT, CIR, HCC, to MET tissues. Arg/Cit correlated significantly with Arg/Orn ratios (r = 0.77; p < 0.0001), and discriminates tumor from non-tumor samples. In addition, the discriminant lactate/glucose ratio we previously found by NMR, also correlated significantly with the Arg levels (r = 0.64; p < 0.0001), and discriminated MET from all other tissues. The results indicated that Arg in MET is higher than other tissue classes, suggesting that, together with the lactate/glucose ratio, it can be considered a further biomarker for HCC-metastases differentiation.     

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN?+?I?+?G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN?+?I?+?G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN?+?I?+?G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN?+?I?+?G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN?+?I?+?G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN?+?I?+?G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN?+?I?+?G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN?+?I?+?G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN?+?I?+?G 0.022 to 0.025) shared the same lineage with T. putitora (TrN?+?I?+?G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.     

The Neuroprotective Effects of α-Iso-cubebene on Dopaminergic Cell Death: Involvement of CREB/Nrf2 Signaling

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

The influence of repetitive thermal stresses on the dominance of reef-building Acropora spp. (Scleractinia) on coral reefs of the Maldive Islands

The distribution and size-age structure of Acropora corals were studied in two Maldivian atolls that differ in their geographic position and sea surface temperature regimes. The frequency and strength of thermal anomalies for the last 2 decades had a significant influence on the abundance, mortality rates, and age structures of acroporid communities. The long-term temperature amplitude was higher and the maxima were more pronounced in the northernmost Ihavandippolu Atoll than those in the equatorial South Huvadhoo Atoll. These differences resulted in a 10.4% mean cover of Acropora at Ihavandippolu Atoll, whereas the Acropora cover in the South Huvadhoo Atoll reached 59.5%. In the northern atoll, the coral mortality rate after the 2010 thermal anomaly was 3 times higher than that in the southern atoll. Younger acroporid colonies (up to 2 years old) dominated the northern atoll reefs, while the southern atoll showed a high proportion of older mature colonies. In both atolls, healthy table colonies of Acropora cytherea with a disk diameter greater than 2 m were observed that apparently survived three thermal anomalies since 1998. The mechanisms of acclimatization of Acropora and the prospects for its dominance in the Maldives under changing environmental conditions are discussed.     

NDRG1 expression is related to the progression and prognosis of gastric cancer patients through modulating proliferation,invasion and cell cycle of gastric cancer cells

N-myc downstream-regulated gene 1 (NDRG1) has been proposed as a tumor suppressor gene in many different types of tumors, but its potential function and corresponding mechanism are not yet fully elucidated. This study aims to detect the possible function of NDRG1 in gastric cancer progression. In this study, 112 paired gastric cancer tissues and corresponding nonmalignant gastric tissues were utilized to identify the differential protein expression of NDRG1 by immunohistochemistry and its clinical significance was analyzed. Furthermore, 49 of 112 paired gastric specimens were used to detect the differential mRNA expression by real-time PCR. The over expression of NDRG1 in human gastric cancer cell line AGS by PcDNA3.1–NDRG1 transfection was utilized to detect the role of NDRG1 in regulating the biological behavior of gastric cancer. NDRG1 expression was significantly decreased in primary gastric cancer tissues, compared with its corresponding nonmalignant gastric tissues (p < 0.05), and its decreased expression was significantly associated with lymph node metastasis (p < 0.01), invasion depth (p < 0.01) and differentiation (p < 0.05). Additionally, the overall survival rate of gastric cancer patients with high expression of NDRG1 was higher than those with low expression during the follow-up period. NDRG1 overexpression suppressed cells proliferation, invasion and induced a G1 cell cycle arrest in gastric cancer. Furthermore, the down-regulation of NDRG1 in gastric cancer metastatic progression was correlated to E-cadherin and MMP-9. Our results verify that NDRG1 acts as a tumor suppressor gene and may play an important role in the metastasis progression and prognosis of gastric cancer.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells

Cystic fibrosis transmembrane conductance regulator (CFTR) acts as a cAMP-dependent chloride channel, has been studied in various types of cells. CFTR is abundantly expressed in vascular smooth muscle cells and closely linked to vascular tone regulation. However, the functional significance of CFTR in basilar vascular smooth muscle cells (BASMCs) remains elusive. Accumulating evidence has shown the direct role of CFTR in cell apoptosis that contributes to several main pathological events in CF, such as inflammation, lung injury and pancreatic insufficiency. We therefore investigated the role of CFTR in BASMC apoptotic process induced by hydrogen peroxide (H₂O₂). We found that H₂O₂-induced cell apoptosis was parallel to a significant decrease in endogenous CFTR protein expression. Silencing CFTR with adenovirus-mediated CFTR specific siRNA further enhanced H₂O₂-induced BASMC injury, mitochondrial cytochrome c release into cytoplasm, cleaved caspase-3 and -9 protein expression and oxidized glutathione levels; while decreased cell viability, the Bcl-2/Bax ratio, mitochondrial membrane potential, total glutathione levels, activities of superoxide dismutase and catalase. The pharmacological activation of CFTR with forskolin produced the opposite effects. These results strongly suggest that CFTR may modulate oxidative stress-related BASMC apoptosis through the cAMP- and mitochondria-dependent pathway and regulating endogenous antioxidant defense system.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

Gene expression profiling of endometrium versus bone marrow-derived mesenchymal stem cells: upregulation of cytokine genes

Postulated Stem/progenitor cells involved in endometrium regeneration are epithelial, mesenchymal, and endothelial. Bone marrow (BM) has been implicated in endometrial stem cells. We aimed at studying gene expression profiling of endometrial mesenchymal stem cells compared to BM MSCS to better understand their nature and functional phenotype. Endometrial tissues were obtained from premenopausal hysterectomies (n = 3), minced and enzymatically digested as well as Normal BM aspirates (n=3). Immunophenotyping, differentiation to mesoderm, and proliferation were studied. The expression profile of 84 genes relevant to mesenchymal stem cells was performed. Fold change calculations were determined with SA Biosciences data analysis software. VEGF, G-CSF, and GM-CSF in cultures supernatants of MSCs were assayed by Luminex immunoassay. Endo MSCs possess properties similar to BM MSCs. Cumulative population doubling was significantly higher in Endo MSCs compared to BM MSCs (p < 0.001). 52 core genes were shared between both generated MSCs including stemness, self-renewal, members of the Notch, TGFB, FGF, and WNT.16 downregulated genes (VCAM, IGF1)and 16 upregulated in Endo MSCs compared to BM (p < 0.05 → fourfolds). They included mostly cytokine and growth factor genes G-CSF, GM-CSF, VWF, IL1b, GDF15, and KDR. VEGF and G-CSF levels were higher in Endo MSCs supernatants (p < 0.0001). Cells sharing MSC and endothelial cell characteristics could be isolated from the human endometrium. Endo MSCs share a core genetic profile with BM MSCs including stemness. They show upregulation of genes involved in vasculogenesis, angiogenesis, cell adhesion, growth proliferation, migration, and differentiation of endothelial cells, all contributing to endometrial function.