Real time changes in the expression of eicosanoid synthesizing enzymes during inflammation.

Immune responses during inflammation involve complex, well-coordinated lipid signaling pathways. Eicosanoids are a class of lipid signaling molecules derived from polyunsaturated fatty acids such as arachidonic acid and constitute a major network that controls inflammation and its subsequent resolution. Arachidonic acid is metabolized by enzymes in three different pathways to form a variety of lipid metabolites that can be either pro- or anti-inflammatory. Therefore, an understanding of the time-dependent gene expression, lipid metabolite profiles and cytokine profiles during the initial inflammatory response is necessary, as it will allow for the design of time-dependent therapeutics. Herein, we investigate the multi-level regulation of this process. After stimulating RAW 264.7 cells, a mouse-derived macrophage cell line commonly used to examine inflammatory responses, we examine the gene expression of 44 relevant lipid metabolizing enzymes from the different eicosanoid synthesizing classes. We also measure the formation of lipid metabolites and production of cytokines at selected time points. Results reveal a dynamic relationship between the time-course of inflammation dependent gene expression of the three eicosanoid synthesizing enzymes.

A comparative study of microbial community and dynamics of Asaia in the brown planthopper from susceptible and resistant rice varieties.

BACKGROUND: The brown planthopper (BPH) is likely the most destructive, piercing and sucking monophagous insect pest of rice that causes substantial economic losses to farmers. Although yeast-like symbionts (YLS) and virus transmission have been observed in the BPH, the bacterial population inhabiting the BPH has received minimal research attention. Labelling BPH-associated bacterial species may shed light on BPH biology and the interaction between the BPH and rice to provide novel approaches for the efficient control of this insect pest. RESULTS: We examined RNA-seq results to identify bacterial populations present in different generations of BPHs maintained on susceptible or resistant rice varieties. Overall, 87 operational taxonomic units (OTUs) were determined from the BPH-F0, F6 and F16 generations. These OTUs had Shannon and Simpson index values of 0.37-0.6 and 0.56-1.19, respectively. The evenness values of 0.7-1.00 showed the vastness of the bacterial diversity recovered from the BPH samples. The results showed high species diversity in the BPHs collected from susceptible rice and a high number of members of unclassified bacteria in the BPHs isolated from resistant rice. We noticed that Proteobacteria OTUs were predominant across all samples. Furthermore, PCR data of Asaia species showed variable DNA amplification across the BPH samples collected from susceptible or resistant varieties. The identification of Asaia in BPH eggs and BPH-egg-infected rice revealed its influence on the interaction between the BPH egg and rice. CONCLUSIONS: The BPHs had clear differences in their microbiomes and in their ability to feed on different rice hosts. These variations could have an essential impact on host adaptation and interaction. These results provide a better understanding of the bacterial diversity and interaction of the microbiome of different generations of BPHs. Furthermore, PCR data of Asaia sp. variation across the BPH samples (isolated from different host genotypes selected from the field and laboratory, including BPH eggs and egg-infected rice tissues), suggest that Asaia could be an important member of the insect microbiome involved in adaptation, its interaction with rice and, most importantly, as a paratransgenic tool for insect control.

Rotational Atherectomy in Acute Coronary Syndrome: A Meta-Analysis.

BACKGROUND: The use of rotational atherectomy (RA) in percutaneous coronary intervention (PCI) of acute coronary syndrome (ACS) is considered relatively contraindicated. There have been several observational studies showing RA use in ACS, however, no systemic studies have been undertaken. We sought to evaluate the feasibility and outcomes of RA PCI in ACS by performing a meta-analysis. METHODS: We searched PUBMED, EMBASE, CINAHL, and Cochrane Central Register of Clinical Trials for any studies that evaluated the role of RA PCI in ACS. The outcomes analyzed were all-cause mortality, cardiac mortality, short and long-term major adverse cardiac events (MACE), procedural complications and cardiac perforations. RESULTS: There was a total of 8 retrospective studies with a total population of 1237 with a median follow up of 23 months. The median age of the included patient was 73. Angiographic success rate was 97.4%. The rate of all-cause mortality and cardiac mortality were 5% (range 1-12%, p < 0.001, I2 = 92%) and 2% (range 0-5%, P = 0.03, I2 = 58%) respectively. In-hospital MACE and long-term MACE were 7% (range 3-13%, p < 0.001, I2 = 87%) and 29% (range 21-37%, p = 0.21, I2 = 34%) respectively. The incidence of total procedural complications was noted to be 7% (range 2-14%, p < 0.001, I2 = 90%). Rate of perforation was 1% (range 0-1%, p = 0.9, I2 = 0%). CONCLUSION: Our results show that RA PCI is feasible in ACS with comparable procedural complications and short-term MACE, but with a higher long-term MACE rate compared to RA PCI in routine cases.

Characterization of gustatory receptor 7 in the brown planthopper reveals functional versatility.

Insect pests consume tastants as their necessary energy and nutrient sources. Gustatory receptors play important roles in insect life and can form within an extremely complicated regulatory network. However, there are still many gustatory genes that have a significant impact on insect physiology, but their functional mechanism is still unknown. Here, we purified and characterized a gustatory receptor (protein) coding gene, NlGr7, from the brown planthopper (BPH) Nilaparvata lugens, which is an important insect pest of rice. Our results revealed that NlGr7 has an active association with various ligands, such as lectins, lipids (phospho- and sphingolipid) and copper. The mass-spectrometry result showed that NlGr7 is a sugar receptor, and NlGr7 is validated by different types of insoluble polysaccharides and a varied range of tastants. Further, we observed that NlGr7-bound ATP hydrolysed on the ATPase activity assay, which indicated that NlGr7 may be associated with important biological functions in the BPH. Furthermore, an injection of NlGr7 (protein), into newly emerged female adults of BPH, showed the reduced vitellogenin in ovary. The important NlGr7 for chemoreception has now been characterized in the BPH. We showed that NlGr7 in the BPH is required for various protein-ligands, as well as protein-sugars interactions, and for regulation of fecundity marker to play crucial roles in this pest. This study will provide valuable information for further functional studies of chemoreception mechanisms in this important agricultural pest.

An Overview of Embryogenesis: External Morphology and Transcriptome Profiling in the Hemipteran Insect Nilaparvata lugens.

During embryogenesis of insects, the morphological and transcriptional changes are important signatures to obtain a better understanding of insect patterning and evolution. The brown planthopper Nilaparvata lugens is a serious insect pest of rice plants, but its embryogenesis has not uncovered. Here, we described embryonic development process of the pest and found it belongs to an intermediate-germ mode. The RNA-seq data from different times (6, 30, 96, and 150 h, after egg laying) of embryogenesis were then analyzed, and a total of 10,895 genes were determined as differentially expressed genes (DEGs) based on pairwise comparisons. Afterward, 1,898 genes, differentially expressed in at least two comparisons of adjacent embryonic stages were divided into 10 clusters using K means cluster analysis (KMCA). Eight-gene modules were established using a weighted gene co-expression network analysis (WGCNA). Gene expression patterns in the different embryonic stages were identified by combining the functional enrichments of the stage-specific clusters and modules, which displayed the expression level and reprogramming of multiple developmental genes during embryogenesis. The "hub" genes at each embryonic stage with possible crucial roles were identified. Notably, we found a "center" set of genes that were related to overall membrane functions and might play important roles in the embryogenesis process. After parental RNAi of the MSTRG.3372, the hub gene, the embryo was observed as abnormal. Furthermore, some homologous genes in classic embryonic development processes and signaling pathways were also involved in embryogenesis of this insect. An improved comprehensive finding of embryogenesis within the N. lugens reveals better information on genetic and genomic studies of embryonic development and might be a potential target for RNAi-based control of this insect pest.

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of GM Proteins in Transgenic Crops/Produce.

Enzyme-linked immunosorbent assays are always being used extensively for the identification of genetically modified protein in transgenic crops/produce. With the advancement in the detection strategies, competitive and sandwich immunoassays are more convenient and sensitive for the GM protein detection and its quantification in transgenic crops. This chapter is focusing on the competitive ELISA including sandwich ELISA for the detection of the GM proteins.

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

Bacterial Community Structure in the Asian Rice Gall Midge Reveals a Varied Microbiome Rich in Proteobacteria.

The Asian rice gall midge (ARGM) has emerged as a model gall forming pest of rice. The ARGM infestation of rice results in failure of panicle formation and economic loss. Understanding the molecular basis of ARGM-rice interactions is very crucial in order to control this devastating pest of rice. The current investigation was devised to identify bacterial communities present in the ARGM and in addition the bacterial diversity in the maggots during their interaction with susceptible or resistant rice varieties. Sequencing of 16S rRNA bacterial gene (V3-V4 region) revealed differences in the microflora of the ARGM maggots feeding on susceptible or resistant rice hosts. Results revealed that Wolbachia was the predominant bacterium in pupae and adults while Pseudomonas was predominant in maggots. Further, we observed that members of proteobacteria were predominant across all the samples. There was high species diversity in maggots isolated from susceptible rice and a high representation of unclassified bacteria in maggots isolated from resistant rice. This is the first study that reports variation of microbiome of the ARGM, based on host phenotype from which it was isolated, and results suggest that these variations could have an important role in host's susceptibility.

Multiple enzymatic activities of ParB/Srx superfamily mediate sexual conflict among conjugative plasmids.

Conjugative plasmids are typically locked in intergenomic and sexual conflicts with co-resident rivals, whose translocation they block using fertility inhibition factors (FINs). We describe here the first crystal structure of an enigmatic FIN Osa deployed by the proteobacterial plasmid pSa. Osa contains a catalytically active version of the ParB/Sulfiredoxin fold with both ATPase and DNase activity, the latter being regulated by an ATP-dependent switch. Using the Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS), a relative of the conjugative T4SS, we demonstrate that catalytically active Osa blocks T-DNA transfer into plants. With a partially reconstituted T4SS in vitro, we show that Osa degrades T-DNA in the T-DNA-VirD2 complex before its translocation. Further, we present evidence for conservation and interplay between ATPase and DNase activities throughout the ParB/Sulfiredoxin fold, using other members of the family, namely P1 ParB and RK2 KorB, which have general functional implications across diverse biological contexts.

Analysis of resistance to Cry1Ac in field-collected pink bollworm, Pectinophora gossypiella (Lepidoptera:Gelechiidae), populations.

High survivorship of pink bollworrm, Pectinophora gossypiella in bolls of Bollgard® cotton hybrids and resistance to Cry1Ac protein, expressed in Bollgard cotton were reported in field-populations collected from the state of Gujarat (western India) in 2010. We have found Cry1Ac-resistance in pink bollworm populations sourced from Bollgard and non-Bt cotton fields in the adjoining states of Maharashtra and Madhya Pradesh in Central India. Further, we observed reduced binding of labeled Cry1Ac protein to receptors localized on the brush-border membrane of pink bollworm larval strains with high tolerance to Cry1Ac. These strains were sourced from Bollgard and conventional cotton fields. A pooled Cry1Ac-resistant strain, further selected on Cry1Ac diet also showed significantly reduced binding to Cry1Ac protein. The reduced binding of Cry1Ac to receptors could be an underlying mechanism for the observed resistance in pink bollworm populations feeding on Bollgard hybrids.

Development of enzyme-linked immunosorbent assay for the detection of Bt protein in transgenic cotton.

Enzyme-linked immunosorbent assays (ELISA) are being used extensively for the identification of Bt-protein in Bt transgenic crops. A sandwich ELISA test is the most preferable immunoassay for the quantification of Bt-protein in transgenic cotton plants. Here, we describe development of sandwich ELISA, employing polyclonal rabbit antibody as a capture antibody and HRP-labeled mouse anti-Bt protein-antibody as a detector antibody.

Host plant induced variation in gut bacteria of Helicoverpa armigera.

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant.

Development of an enzyme linked immunosorbant assay for the detection of Cry2Ab Protein in transgenic plants.

Immunoassays' a sensitive technique has a wide application for detection of antigen, especially for the detection of growing genetically modified crops, which are underwent field trial. Here, we have developed for the quantitative detection of Cry2Ab protein expressed in GM crops. A Cry2Ab-rabbit-IgG act as capture antibody and Cry2Ab mouse monoclonal antibody behave as a detecting antibody, which is employed in sandwich ELISA. A 2% polyvinylpyrrollidone and 1% dithiothritol was utilized in protein extraction from cotton seed to avert interfering agents. The developed assay was validated with GM cotton samples, limit of detection was found 1 pg/ml and range to a limit of quantification 16 pg/ml. The developed immunoassay does not show any cross reactivity with other non target GM proteins like Cry1Ab, Cry1Ac, EPSPS, Vip.