Codon optimization of <Emphasis Type="Italic">cry1Ab</Emphasis> gene for hyper expression in plant organelles

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.     

Rhizobacterial potential to alter auxin content and growth of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Potential of non-symbiotic plant growth promoting rhizobacteria (PGPR) to influence the endogenous indole-3-acetic acid (IAA) content and growth of Vigna radiata (L.) was evaluated. The bacterial strains used belonged to Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera. All strains were able to produce IAA (1.16–8.22 μg ml⁻¹) in the presence of 1,000 μg ml⁻¹ of l-tryptophan as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. However, strains exhibited variable results for other growth promoting traits such as phosphate solubilization and siderophore or hydrogen cyanide production. Bacterial IAA production showed significant positive correlation with endogenous IAA content of roots (r = 0.969; P = 0.01) and leaves (r = 0.905; P = 0.01) under axenic conditions. Bacterization of V. radiata seeds significantly enhanced shoot length (up to 48.10%) and shoot fresh biomass (up to 43.80%) under fully axenic conditions. Bacterial strains applied under wire-house conditions also improved shoot length, number of pods, and grain weight up to 58, 65, and 17.15% respectively, over control. Hence, free living (non-symbiotic) PGPR have the ability to influence endogenous IAA content and growth of leguminous plants.     

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

Salicylic acid-mediated changes in photosynthesis,nutrients content and antioxidant metabolism in two mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in salt tolerance

Mustard (Brassica juncea L.) cultivars Alankar (salt-tolerant) and PBM16 (salt-sensitive) plants were grown with 50 mM NaCl and were sprayed with 0.1, 0.5, and 1.0 mM salicylic acid (SA) to study the physiological processes determining salt tolerance and to observe the influence of SA application on the alleviation of NaCl-induced adverse effects. The content of leaf Na⁺, Cl⁻, H₂O₂, TBARS, and electrolyte leakage and the activity of SOD were higher in PBM16 than Alankar. In contrast, nutrients content, activity of APX and GR, glutathione content, photosynthetic and growth characteristics were higher in Alankar. Treatment of 50 mM NaCl resulted in increase of Na⁺ and Cl⁻, oxidative stress, activity of antioxidant enzymes and glutathione content, while nutrients content, photosynthetic, and growth characteristics decreased in both the cultivars. Application of 0.5 mM SA alleviated the negative effects of 50 mM NaCl maximally, but 1.0 mM SA proved inhibitory. The effect of SA was more conspicuous in Alankar than PBM16. It is concluded that the higher tolerance of Alankar was due to its lower leaf Na⁺ and Cl⁻ content, higher nutrients content, and efficient antioxidant metabolism. The application of 0.5 mM SA substantially alleviated salt-induced adverse effects in Alankar.     

Transgenerational phenotypic plasticity of diapause induction and related fitness cost in a commercial strain of the parasitoid Aphidius ervi Haliday

Diapause is an adaptation that insects have evolved to synchronize their life cycle with that of seasonal climatic changes and resources availability. However, cues for its induction are not always clear and, in some cases, a maternal effect may be involved. At the population level, just a part of the individuals may exhibit diapause with important consequences in terms of winter survival. Moreover, clear indicators of diapause state are difficult to identify. Diapause induction was thus investigated in the aphid parasitoid species Aphidius ervi Haliday (Hymenoptera: Braconidae) developing in the aphid Sitobion avenae (Hemiptera: Aphididae) at four crossed photothermal regimes (16 °C and 8 °C, 16:8 h L:D and 8:16 h L:D), and during 2 successive generations. We analyzed the reliability of changes in mummy color to assess for the diapausing state compared to dissections, and we measured parasitoid morphological and physiological traits. We observed that the proportion of dark brown mummies increased after one generation under low photothermal regime compared to other regimes. No diapause was recorded at 16 °C, 16:8 h L:D, while we observed 16.2% and 67.5% diapause incidence at 8 °C, 8:16 h L:D, at 1st and 2nd generation, respectively. Diapause induction is thus increased by short day-length conditions and low temperatures as well as by maternal effects. All parasitoid life-history traits (weight, size, fat content, water content, egg-load, and longevity) were affected by the photothermal regime and/or the generation. These results raise new questions on the environmental thresholds needed to induce diapause and on survival and adaptation potential of commercially available parasitoid strains in different environments.     

Genetic analysis of NaCl tolerance in tomato

We attempted to find the suitable parents for the development of tomato hybrids for high salt soils by exploiting combining ability, gene action and heterosis. Six salt-tolerant and three salt-intolerant genotypes, along with their 18 F1 crosses, were evaluated at seedling stage under 10 and 15 dS/m (NaCl) salinity stress, compared to the control level of salinity. The experiment was laid out based on a two-way complete randomized design factorial arrangement with two replications; data on root and shoot length, fresh and dry weights, leaf area, plant length, Na(+), K(+) and K(+)/Na(+) concentrations were recorded. There was significant variation within genotypes, lines, testers, crosses, and line × tester interaction for all plant characters studied under normal and two salinity levels. Estimates of combining ability indicated that under low (10 dS/m) and high (15 dS/m) salinities, line BL1176 and tester LO2875 showed significant GCA effects for most of the traits studied. The cross-combinations 6233 × LO2875, CLN2498A x LO2875 and BL1176 × 17902 showed highest SCA values for most of the characters under 10 and 15 dS/m, respectively. Potence ratio showed that under low and high salinities, all the traits showed over dominant type of gene action except leaf area and K(+) concentration (in 10 dS/m) and shoot length, and leaf area (in 15 dS/m). The highest heterosis for most of the parameters was observed in cross-combinations BL1176 × LO2875 and CLN2498A x LO2875.     

The potential influence of common viral infections diagnosed during hospitalization among critically ill patients in the United States

Viruses are the most common source of infection among immunocompetent individuals, yet they are not considered a clinically meaningful risk factor among the critically ill. This work examines the association of viral infections diagnosed during the hospital stay or not documented as present on admission to the outcomes of ICU patients with no evidence of immunosuppression on admission. This is a population-based retrospective cohort study of University HealthSystem Consortium (UHC) academic centers in the U.S. from the years 2006 to 2009. The UHC is an alliance of over 90% of the non-profit academic medical centers in the U.S. A total of 209,695 critically ill patients were used in this analysis. Eight hospital complications were examined. Patients were grouped into four cohorts: absence of infection, bacterial infection only, viral infection only, and bacterial and viral infection during same hospital admission. Viral infections diagnosed during hospitalization significantly increased the risk of all complications. There was also a seasonal pattern for viral infections. Specific viruses associated with poor outcomes included influenza, RSV, CMV, and HSV. Patients who had both viral and bacterial infections during the same hospitalization had the greatest risk of mortality RR 6.58, 95% CI (5.47, 7.91); multi-organ failure RR 8.25, 95% CI (7.50, 9.07); and septic shock RR 271.2, 95% CI (188.0, 391.3). Viral infections may play a significant yet unrecognized role in the outcomes of ICU patients. They may serve as biological markers or play an active role in the development of certain adverse complications by interacting with coincident bacterial infection.     

Sphingomyelin synthase 2 (SMS2) deficiency attenuates LPS-induced lung injury

Sphingomyelin synthase (SMS) catalyzes the synthesis of sphingomyelin (SM) and is required for maintenance of plasma membrane microdomain fluidity. Of the two isoforms of mammalian SMS, SMS1 is mostly present in the trans-Golgi apparatus, whereas SMS2 is predominantly found at the plasma membrane. SMS2 has a role in receptor mediated response to inflammation in macrophages, however, the role of SMS2 in vascular permeability, pulmonary edema, and lung injury have not been investigated. To define the role of SMS activation in lung injury, we utilized a lipopolysaccharide (LPS)-induced lung edema model. SMS activity was measured and correlated with the severity of lung injury. Within 4 h of LPS treatment, SMS activity was increased significantly and remained upregulated up to 24 h. Comparison of LPS-induced lung injury in SMS2 knockout (SMS2(-/-)) and wild-type littermate control mice showed that inflammation, cytokine induction, and lung injury were significantly inhibited in SMS2(-/-) mice. Our results suggest that a deficiency of SMS2 can diminish the extent of pulmonary edema and lung injury. Furthermore, we show that depletion of SMS2 was sufficient to decrease MAP kinase-JNK activation, severity of LPS-induced pulmonary neutrophil influx, and inflammation, suggesting a novel role of SMS2 activation in lung injury.