The molecular mechanism of WRINKLED1 transcription factor regulating oil accumulation in developing seeds of castor bean.

The transcription factor WRINKLED1 (WRI1), a member of AP2 gene family that contain typical AP2 domains, has been considered as a master regulator regulating oil biosynthesis in oilseeds. However, the regulatory mechanism of RcWRI1 in regulating oil accumulation during seed development has not been clearly addressed. Castor bean (Ricinus communis) is one of the most important non-edible oil crops and its seed oils are rich in hydroxy fatty acids, widely applied in industry. In this study, based on castor bean reference genome, three RcWRIs genes (RcWRI1, RcWRI2 and RcWRI3) were identified and the expressed association of RcWRI1 with oil accumulation were determined. Heterologous transformation of RcWRI1 significantly increased oil content in tobacco leaf, confirming that RcWRI1 activate lipid biosynthesis pathway. Using DNA Affinity Purification sequencing (DAP-seq) technology, we confirmed RcWRI1 binding with Transcription Start Site of genes and identified 7961 WRI1-binding candidate genes. Functionally, these identified genes were mainly involved in diverse metabolism pathways (including lipid biosynthesis). Three cis-elements AW-box ([CnTnG](n)7[CG]) and AW-boxes like ([GnAnC](n)6[GC]/[GnAnC](n)7[G]) bound with RcWRI1 were identified. Co-expression network analysis of RcWRI1 further found that RcWRI1 might be widely involved in biosynthesis of storage materials during seed development. In particular, yeast one hybrid experiments found that both AP2 domains within RcWRI1 were required in binding targeted genes. These results not only provide new evidence to understand the regulatory mechanism of RcWRI1 in regulation of oil accumulation during castor bean seed development, but also give candidate gene resource for subsequent genetic improvement toward increasing oil content in oilseed crops.

Development of an efficient chromatin immunoprecipitation method to investigate protein-DNA interaction in oleaginous castor bean seeds.

Chromatin immunoprecipitation (ChIP) is usually a reliable technique to find the binding sites of a transcription factor. In the current study, we developed a suitable ChIP method using developing castor bean seeds. A castor bean seed with large and persistent endosperm contains high amounts of storage lipids (ca. 50-60%) and is often considered as a model material to studying seed biology. In oleaginous seeds, due to the rich oils which could seriously affect immunoprecipitation and DNA isolation, it is often difficult to carry out a successful ChIP experiment. Thus, the development of an efficient ChIP method for oleaginous seeds is required. In this study, we modified different steps, including tissue preparation for cross-linking, chromatin washing, sonication and immunoprecipitation of other existing methods. As exemplified by the targeted gene identification of a master regulator WRI1, which regulates fatty acid biosynthesis, we found that the improved ChIP method worked well. We analyzed percentage input and fold changes of the ChIPed DNA. We also made successful ChIP-seq libraries using this method. This method provides a technical support not only for use on castor bean seeds; it might be used equally to analyze protein-DNA interaction in vivo in other oleaginous seeds.

Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh.

OBJECTIVE: Better methods are needed for the accurate detection of child tuberculosis (TB). This study compared different laboratory tests and evaluated IS6110 PCR for the detection of Mycobacterium tuberculosis (MTB) among clinically diagnosed child TB patients. METHODS: A total of 102 paediatric patients (<15 years old) with clinically diagnosed TB were enrolled in this study. The patients were admitted to the icddr,b hospital in Dhaka between 2003 and 2005. Sputum/gastric lavage samples were collected for smear microscopy, culture (solid/Lowenstein-Jensen medium and liquid/MGIT), and IS6110 PCR testing. The sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of smear microscopy and PCR were compared to the two culture methods. RESULTS: Three patients were positive on smear microscopy (2.9%). MTB was detected by conventional culture in 15.7% (16/102), liquid culture in 14% (14/100), and IS6110 PCR in 61.8% (63/102). PCR detected an additional 45 patients who were undetected with the three other tests. Compared to conventional and liquid culture, respectively, smear microscopy showed sensitivity of 18.8% and 21.4%, specificity of 100% individually, PPV of 100% individually, and NPV of 86.9% and 88.7%, whereas PCR had sensitivity of 87.5% and 92.9%, specificity of 43% individually, PPV of 22.2% and 21%, and NPV of 94.9% and 97.4%. CONCLUSIONS: PCR can be useful compared to smear microscopy and culture methods and is applicable as a rapid screening test for child TB. A larger scale study is required to determine its diagnostic efficacy in improving the detection of child TB in the presence and absence of severe malnutrition.

Transcriptomic analyses reveal complex and interconnected sucrose signaling cascades in developing seeds of castor bean.

Seeds are highly specific organs that strongly sink sucrose resources from leaf and stem tissues to trigger seed metabolism and development. In particular, for heterotrophic non-green seeds, the potential molecular mechanism underlying sucrose-driven seed development remains unanswered. Castor bean (Ricinus communis L.), a typical non-green seed, has been considered as a model plant for seed biology study in dicotyledonous plants due to its heterotrophic seeds with persistent endosperms. In the present study, the fast-developing castor bean seeds were treated with exogenous sucrose and mannitol for four hours. The global transcriptomic data were obtained by high-throughput RNA-seq technique, resulting in 468 differentially expressed genes (DGEs). Further analyses revealed that sucrose functioned as both metabolic substrates and signal molecules. Specifically, 73 DGEs involved in carbohydrate and nitrogen metabolism, 42 differentially expressed transcription factors, and 35 DGEs involved in diverse signaling pathways such as auxin, brassinosteroid, ethelyene, cytokinin, gibberellin, and calcium signals, were identified, suggesting that the sucrose signaling pathway might have complex and multi-connected cross-talks with other signals to regulate castor bean seed development. Taken together, this study provides novel data to improve understanding of the potential molecular mechanisms of sucrose in regulating non-green seed development and storage reservoir accumulation during seed development.

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Why Bangladeshi nurses avoid 'nursing': social and structural factors on hospital wards in Bangladesh.

In response to concerns that nurses spend less than 6% of their time on direct patient care, this study explored factors that influence nurses' behaviour in the provision of 'hands on' care in hospitals in Bangladesh. Through in-depth interviews with female nurses and patients and their co-workers in six hospitals, we identified conflicts between the inherited British model of nursing and Bangladeshi societal norms. This was most evident in the areas of night duty, contact with strangers, and involvement in 'dirty' work. The public was said to associate nursing activities with commercial sex work. As a consequence, their value on the 'bride market' decreases. To minimise the stigma associated with their profession, nurses in government hospitals distance themselves from patients, using nurse surrogates in the form of patients' relatives and hospital support workers to carry out their work. These adaptations are supported and sustained through unofficial activities developed over time within hospitals. In contrast nurses in NGO hospitals give more direct patient care themselves and do not rely on carers as much because of tight supervision and limited visitor hours. Initiatives undertaken to improve the quality of patient care, such as enlarging the nursing workforce or providing clinical instruction, which do not take into account the prevailing culture in hospitals and social conflicts faced by nurses, are unlikely to succeed. Fundamental decisions on how to care for the sick in Bangladesh are required. If the present nursing curriculum is followed, adequate supplies, supervision and accountability are prerequisites for its implementation.