Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

Taxonomic review and phylogenetic inference elucidate the evolutionary history of Mesozoic Procercopidae,with new data from the Cretaceous Jehol Biota of NE China (Hemiptera,Cicadomorpha)

The Mesozoic family Procercopidae is widely treated as the ancient group of Cercopoidea and a transitional unit to recent lineages, but its evolution and diversity are vague due to fragmentary fossil record and confusing taxonomic history. Herein, an extensive taxonomic review of Procercopidae is presented and some new fossils are reported from the Lower Cretaceous Yixian Formation of NE China. As a result, Chengdecercopis Hong, 1983 is transferred from Procercopidae to Sinoalidae; Procercopis longipennis Becker-Migdisova, 1962 and P shawanensis Zhang, Wang and Zhang, 2003 are transferred to Procercopina Martynov, 1937, resulting in Procercopina longipennis (Becker-Migdisova, 1962), comb. n. and P shawanensis (Zhang, Wang and Zhang, 2003), comb. n.; Luanpingia senjituensis Hong, 1984 is transferred to Stellularis Chen, Yao and Ren, 2015, leading to Stellulari senjituensis (Hong, 1984), comb. n.; Anthoscytina macula Hu, Yao and Ren, 2014 is transferred to Sinocercopis Hong, 1982, and Sunoscytinopteris (Scytinopteridae) and Cathaycixius (Cixiidae) are treated as junior homonym names of Sinocercopis, leading to Sinocercopis macula (Hu, Yao and Ren, 2014), comb. n., S lushangfenensis (Hong, 1984), comb. n., S pustulosis (Ren, 1995), comb. n., and S trinervis (Ren, 1995), comb. n. Additionally, two new species are erected: Stellularis bineuris Chen and Wang, sp. n. and S minutus Chen and Wang, sp. n. Our cladistic analysis based on wing (tegmen and hind wing) characteristics recovers the high-level relationships within Cercopoidea: Sinoalidae + (Procercopidae + (Cercopionidae + modern cercopoids)). Within the family Procercopidae, the cladistic analysis reveals that the Middle to Late Jurassic Titanocercopis and Jurocercopis and the Cretaceous Cretocercopis occupy the basal position, and a gradual change in wing venation can be recognized from the Early Jurassic Procercopis and Procercopina to the Jurassic Anthoscytina, and then to the Cretaceous Stellularis and Sinocercopis. The two Cretaceous genera, sharing wing traits with extant cercopoids, likely represent transitional forms between Procercopidae and recent Cercopoidea; however, they are very similar to their Jurassic relatives in body structures, suggesting it is applicable to attribute them to Procercopidae. Furthermore, our analysis suggests that the extinction of Procercopidae and the origin and early diversification of modern Cercopoidea approximately coincided with the rise and explosive radiation of angiosperms in the late Early Cretaceous and onwards.     

An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.     

Prevalence of Cataract Surgery and Visual Outcomes in Indian Immigrants in Singapore: The Singapore Indian Eye Study

Objective

To determine the prevalence of cataract surgery and factors associated with post-surgical visual outcomes in migrant Indians living in Singapore.

Research Design and Methods

We conducted a population-based study in 3,400 Indian immigrants residing in Singapore−the Singapore Indian Eye Study (SINDI). All participants underwent comprehensive medical eye examination and a standardized interview. Post-operative visual impairment (VI) was defined as best-corrected or presenting visual acuity (BCVA or PVA) of 20/60 or worse.

Results

The age- and gender-standardized prevalence of cataract surgery was 9.7% (95% confidence interval [CI]: 8.9%, 10.7%) in Singapore resident Indians. Post-operative VI defined by BCVA occurred in 10.9% eyes (87/795). The main causes of post-operative VI were diabetic retinopathy (20.7%), posterior capsular opacification (18.4%), and age-related macular degeneration (12.6%). Undercorrected refractive error doubled the prevalence of post-operative VI when PVA was used.

Conclusions

The rate of cataract surgery is about 10% in Indian residents in Singapore. Socioeconomic variables and migration had no significant impact on the prevalence of cataract surgery. Diabetic retinopathy was a major cause of post-operative VI in migrant Indians living in Singapore. Uncorrected postoperative refractive error remains an efficient way to improve vision.     

Reprogramming Substrate and Catalytic Promiscuity of Tryptophan Prenyltransferases

Prenylation is a process widely prevalent in primary and secondary metabolism, contributing to functionality and chemical diversity in natural systems. Due to their high regio- and chemoselectivities, prenyltransferases are also valuable tools for creation of new compounds by chemoenzymatic synthesis and synthetic biology. Over the last ten years, biochemical and structural investigations shed light on the mechanism and key residues that control the catalytic process, but to date crucial information on how certain prenyltransferases control regioselectivity and chemoselectivity is still lacking. Here, we advance a general understanding of the enzyme family by contributing the first structure of a tryptophan C5-prenyltransferase 5-DMATS. Additinally, the structure of a bacterial tryptophan C6-prenyltransferase 6-DMATS was solved. Analysis and comparison of both substrate-bound complexes led to the identification of key residues for catalysis. Next, site-directed mutagenesis was successfully implemented to not only modify the prenyl donor specificity but also to redirect the prenylation, thereby switching the regioselectivity of 6-DMATS to that of 5-DMATS. The general strategy of structure-guided protein engineering should be applicable to other related prenyltransferases, thus enabling the production of novel prenylated compounds.     

CpG or IFN-α are more potent adjuvants than GM-CSF to promote anti-tumor immunity following idiotype vaccine in multiple myeloma

Idiotype (Id) protein in combination with GM-CSF has been used as vaccines for immunotherapy of patients with myeloma and B-cell tumors and the results have been disappointing. To search for better immune adjuvants to improve the efficacy of Id-based immunotherapy in myeloma, we evaluated and compared the efficacy of vaccination of Id protein in combination with CpG or IFN-α, or GM-CSF as a control, in the 5TGM1 myeloma mouse model. Our results showed that Id vaccine combined with CpG or IFN-α, but not GM-CSF, not only efficiently protected mice from developing myeloma but also eradicated established myeloma. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-Id antibodies, and cellular immune responses including Id- and myeloma-specific CD8⁺ cytotoxic T lymphocytes (CTLs), CD4⁺ type-1 T-helper (Th1) cells and memory T cells in mice receiving Id vaccine combined with CpG or IFN-α. Furthermore, Id vaccine combined with CpG or IFN-α induced Id- and tumor-specific memory immune responses that protected surviving mice from tumor rechallenge. Thus, our study clearly shows that CpG or IFN-α are better immune adjuvants than GM-CSF. This information will be important for improving the strategies of Id-based immunotherapy for patients with myeloma and other B-cell tumors.     

Enantioselective determination of dencichine in rabbit plasma by high-performance liquid chromatography-electrospray mass spectrometry

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C(18) cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-L-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect D-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for D-dencichine and 8 ng/ml for L-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 microg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.     

Ricin A chain reaches the endoplasmic reticulum after endocytosis

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.     

Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct alpha-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.