Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-à-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage

Expression of Multiple Resistance Genes Enhances Tolerance to Environmental Stressors in Transgenic Poplar (Populus × euramericana ‘Guariento’)

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana ‘Guariento’) harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone

Shock waves can enhance bacterial transformation with plasmid DNA

Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are strong perturbations in aerodynamics that propagate at supersonic speeds independent of the wave amplitude. Such disturbances occur in steady transonic or supersonic flow during explosions, lightening strokes and contact surfaces in laboratory devices. The typical thickness of the shock front in air is $~10^{\mathrm{-7}}m$, very small compared to other characteristic lengths in fluid flow. Physically, the occurrence of shock waves is always characterized in a fluid flow by instantaneous changes in pressure, velocity and temperature

Development of transgenic pigeonpea (Cajanus cajan. L Millsp) overexpressing citrate synthase gene for high phosphorus uptake

493-501Plants have developed several adaptive strategies to enhance the availability and uptake of phosphorus (P) from the soil under conditions of P deficiency. Exudation of organic acids like citrate is one of the important strategies. In this study, we developed transgenic pigeonpea (Cajanus cajan) over-expressing Dacus carota citrate synthase (DcCs) gene to increase the synthesis and exudation of citrate. Transgenic plants were generated through agro bacterium mediated in-planta transformation technique. Integration and expression of the transgene was confirmed by genomic Southern and RT-PCR analysis. We observed that the transgenic lines had more tissue P and chlorophyll content, and also citrate synthase content higher in the roots. Further, transgenic lines had more vigorous root system both under P sufficient and deficient conditions with more lateral roots and root hairs under P deficient conditions. We conclude that the transgenic pigeonpea plants have the capacity to acquire more P under P deficient conditions

Isolation of endophytic bacteria producing the anti-cancer alkaloid camptothecine from Miquelia dentata Bedd. (Icacinaceae)

Camptothecine (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this activity, several semi-synthetic derivatives of CPT are in clinical use against ovarian and small lung cancers. Together with its derivatives, CPT is the third largest anti-cancer drug in the world market. CPT is produced by several plant species belonging to the Asterid clade. In the recent past, several studies have reported the production of CPT by endophytic fungal associates of some of these plant species. In this paper, we report the production of CPT by endophytic bacteria isolated from Miquelia dentata Bedd. (Icacinaceae). Besides CPT, the bacteria also produced 9-methoxy CPT (9-MeO-CPT), in culture, independent of the host tissue. The chemical nature of CPT and 9-MeO-CPT was determined by LC–MS and ESI-MS/MS analysis, and was shown to be similar to that produced by the host tissue. One of the bacterial isolates examined, showed indications of attenuation of CPT production through sub-culture. This is the first report of production of CPT by endophytic bacteria. The identity of the bacteria was ascertained by Gram staining and 16s rRNA sequencing. We discuss the possible mechanisms that might be involved in the synthesis of CPT by endophytic bacteria

Thermotolerance of fungal endophytes isolated from plants adapted to the Thar Desert, India

A total of 507 endophytic fungal isolates belonging to 82 operational taxonomic units (OTUs) were isolated from fifteen plant species naturally occurring in the Thar Desert, Rajasthan, India. Aspergillus, Alternaria, Chaetomium, Penicillium and Nigrospora were among the dominant fungal genera. Thermotolerance of the isolates was evaluated by culturing the fungi at 40 °C and 45 °C in shake flasks. Growth of nine OTUs (ACJ-2, ACJ-5, ACL-2, ACT-2, ACT-3, LAS-4, LAS-6, SAP-3 and SAP-6) was unaffected at 40 °C. Of these, six endophytes, namely, ACJ-2, ACJ-5 (Aspergillus flavus), SAP-3 (Aspergillus sp.), SAP-6, LAS-4 (Aspergillus sp.) and LAS-6 (Chaetomium sp.), were tolerant to as high as 45 °C. Rest of the OTUs did not survive culture temperatures beyond 35 °C. We evaluated the ability of one thermotolerant endophyte, LAS-6 (Chaetomium sp.) to confer high temperature tolerance and three OTUs, namely, LAS-4 (Aspergillus sp.), SAP-3 (Aspergillus sp.) and SAP-6 to confer drought tolerance to a rice cultivar, IR-64, at early seedling stage. Seedlings treated with LAS-6 (Chaetomium sp.) showed a higher survival percentage as well as maintained a significantly higher shoot and root growth under high temperature stress compared to seedlings not treated with the fungus. Under drought stress, endophyte treated seedlings maintained a significantly higher root growth compared to untreated seedlings. The identified thermotolerant fungal sources could be potentially useful in alleviating abiotic stress in agriculturally important crops

Role of endophytes in early seedling growth of plants: a test using systemic fungicide seed treatment

Systemic fungicide seed treatments are routinely used in conventional agriculture to control soil and seedborne diseases, but little is known about their unintended adverse effects on non-target beneficial fungal endophytes that are known to be involved in plant growth and development. This study evaluated the seed treatment effect of a broad spectrum systemic fungicide, carbendazim (bavistin) on symbiotic association of fungal endophytes in rice and on early seedling growth of rice, green gram, soybean, and cowpea. Seeds were surface sterilized with sodium hypochlorite followed by 0.2% bavistin treatment. Growth of fungal endophytes was significantly affected by the seed treatment with fungicide in rice seedlings, while shoot and root growth was suppressed in all the crops. Quantitative real time PCR showed that the level of expression of two basal transcriptional regulator genes, OsBTF3 and OsNFYC1 that are required for seed germination and seedling growth significantly decreased in bavistin treated rice seedlings. Re-inoculation of consortia of fungal endophytes onto bavistin treated rice seedlings significantly recovered seedling growth and development. These results suggest that fungicide treatment of seeds affects early seedling growth and has negative impact on beneficial fungal endophytes that are involved in plant growth and development. This study provides information on possible ill effects of fungicide on beneficial fungal endophytes that play key roles in early seedling growth of plants and also open up the prospect to additional research on different crops in vitro and field conditions to determine the consequences of fungicide effects and optimise fungicide application strategies to develop sustainable disease control methods