GenoChemetic strategy for derivatization of the violacein natural product scaffold

H.E.L. was supported by an Imperial College President’s Ph.D. Scholarship. We thank UKRI EPSRC (EP/K038648/1, EP/L011573/1 to P.S.F.) and the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 614779 GenoChemetics to R.J.M.G.) for funding. A.M.C.O. receives funding from EPSRC CRITICAT, EP/L016419/1.Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.Publisher PDFPeer reviewe

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology

Immature embryo: A useful tool for oil palm ( Elaeis guineensis Jacq.) genetic transformation studies

Oil palm ( Elaeis guineensis Jacq.) is the highest yielding oil-bearing crop. However, being a perennial crop, genetic improvement of oil palm is extremely slow. Indeed, compared to other annual oil crops such as soybean and rapeseed, genetic manipulations remained less important. Therefore, to remain competitive, oil palm growers and breeders need new and novel approaches. In this report, the potential of immature embryos (IE) as a useful tool for oil palm genetic transformation studies was evaluated. It was evident that IEs were amenable to both direct and Agrobacterium-mediated gene transfer. Due to the abundant supply of IE, optimization of biolistic and Agrobacterium-mediated gene transfer into IEs were easily carried out. Transient transformation frequencies were comparable to other plant systems reported, with as high as 97.4% recorded for biolistic and 64.4% for Agrobacterium-mediated gene transfer. Like most moncots, oil palm tissues were less sensitive to kanamycin, geneticin and chloramphenicol. Instead, both hygromycin and phosphinotrycin were toxic 20 mg/l, making both suitable candidates for selecting putative transformants. IEs were also more responsive to in vitro manipulations as compared to other explants such as leaf and root tissues. Rapid in vitro response to callusing and embryogenesis or rapid and highly efficient direct germination resulted in a shorter culture period. This would minimize the production of abnormal clonal palms, which has been associated to chromosomal aberration due to prolonged time in culture. In addition, IEs also allows rapid and direct introduction of elite genes into breeding programs and in biclonal seed production

Germline breast cancer susceptibility genes, tumor characteristics, and survival.

BACKGROUND: Mutations in certain genes are known to increase breast cancer risk. We study the relevance of rare protein-truncating variants (PTVs) that may result in loss-of-function in breast cancer susceptibility genes on tumor characteristics and survival in 8852 breast cancer patients of Asian descent. METHODS: Gene panel sequencing was performed for 34 known or suspected breast cancer predisposition genes, of which nine genes (ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, and TP53) were associated with breast cancer risk. Associations between PTV carriership in one or more genes and tumor characteristics were examined using multinomial logistic regression. Ten-year overall survival was estimated using Cox regression models in 6477 breast cancer patients after excluding older patients (≥75years) and stage 0 and IV disease. RESULTS: PTV9genes carriership (n = 690) was significantly associated (p < 0.001) with more aggressive tumor characteristics including high grade (poorly vs well-differentiated, odds ratio [95% confidence interval] 3.48 [2.35-5.17], moderately vs well-differentiated 2.33 [1.56-3.49]), as well as luminal B [HER-] and triple-negative subtypes (vs luminal A 2.15 [1.58-2.92] and 2.85 [2.17-3.73], respectively), adjusted for age at diagnosis, study, and ethnicity. Associations with grade and luminal B [HER2-] subtype remained significant after excluding BRCA1/2 carriers. PTV25genes carriership (n = 289, excluding carriers of the nine genes associated with breast cancer) was not associated with tumor characteristics. However, PTV25genes carriership, but not PTV9genes carriership, was suggested to be associated with worse 10-year overall survival (hazard ratio [CI] 1.63 [1.16-2.28]). CONCLUSIONS: PTV9genes carriership is associated with more aggressive tumors. Variants in other genes might be associated with the survival of breast cancer patients. The finding that PTV carriership is not just associated with higher breast cancer risk, but also more severe and fatal forms of the disease, suggests that genetic testing has the potential to provide additional health information and help healthy individuals make screening decisions

Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.

Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed. Cell Rep 2015 Jul 14; 12(2):272-285

A comparison of clinical characteristics of older adults treated with antidepressants in general and psychiatric hospitals in Asia

Abstract Aim: This study compared the demographics, clinical characteristics, and antidepressant prescription patterns between Asian patients aged 50 years and older attending psychiatric hospitals and those attending general hospitals. Methods: In total, 955 patients (604 in general hospitals, 351 in psychiatric hospitals) aged 50 years or older treated with antidepressants in 10 Asian countries and territories were examined. Patients' demographics, clinical features, and prescriptions of psychotropic drugs were recorded using a standardized protocol and data collection procedure. Results: Binary logistic regression revealed that high income and diagnosis of schizophrenia were independently associated with psychiatric hospital treatment, whereas outpatient care, diagnosis of anxiety disorders, and multiple major medical conditions were independently associated with general hospital treatment. In addition, tetracyclic and noradrenergic and specific serotonergic antidepressants were more likely to be prescribed in general hospitals. Conclusion: Older adults treated with antidepressants showed different demographic and clinical features between general hospitals and psychiatric hospitals in Asia

Clinical Use of Mood Stabilizers With Antidepressants in Asia Report From the Research on Asian Psychotropic Prescription Patterns for Antidepressants (REAP-AD) Projects in 2004 and 2013

Abstract Objective: As most reports concerning treatment with combinations of mood stabilizer (MS) with antidepressant (AD) drugs are based in the West, we surveyed characteristics of such cotreatment in 42 sites caring for the mentally ill in 10 Asian countries. Methods: This cross-sectional, pharmacoepidemiologic study used 2004 and 2013 data from the REAP-AD (Research Study on Asian Psychotropic Prescription Patterns for Antidepressants) to evaluate the rates and doses of MSs given with ADs and associated factors in 4164 psychiatric patients, using standard bivariate methods followed by multivariable logistic regression modeling. Results: Use of MS + AD increased by 104% (5.5% to 11.2%) between 2004 and 2013 and was much more associated with diagnosis of bipolar disorder than major depression or anxiety disorder, as well as with hospitalization > outpatient care, psychiatric > general-medical programs, and young age (all P < 0.001), but not with country, sex, or AD dose. Conclusions: The findings provide a broad picture of contemporary use of MSs with ADs in Asia, support predictions that such treatment increased in recent years, and was associated with diagnosis of bipolar disorder, treatment in inpatient and psychiatric settings, and younger age

Polygenic risk scores for prediction of breast cancer risk in Asian populations.

PURPOSE: Non-European populations are under-represented in genetics studies, hindering clinical implementation of breast cancer polygenic risk scores (PRSs). We aimed to develop PRSs using the largest available studies of Asian ancestry and to assess the transferability of PRS across ethnic subgroups. METHODS: The development data set comprised 138,309 women from 17 case-control studies. PRSs were generated using a clumping and thresholding method, lasso penalized regression, an Empirical Bayes approach, a Bayesian polygenic prediction approach, or linear combinations of multiple PRSs. These PRSs were evaluated in 89,898 women from 3 prospective studies (1592 incident cases). RESULTS: The best performing PRS (genome-wide set of single-nucleotide variations [formerly single-nucleotide polymorphism]) had a hazard ratio per unit SD of 1.62 (95% CI = 1.46-1.80) and an area under the receiver operating curve of 0.635 (95% CI = 0.622-0.649). Combined Asian and European PRSs (333 single-nucleotide variations) had a hazard ratio per SD of 1.53 (95% CI = 1.37-1.71) and an area under the receiver operating curve of 0.621 (95% CI = 0.608-0.635). The distribution of the latter PRS was different across ethnic subgroups, confirming the importance of population-specific calibration for valid estimation of breast cancer risk. CONCLUSION: PRSs developed in this study, from association data from multiple ancestries, can enhance risk stratification for women of Asian ancestry

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms