Improving simvastatin bioconversion in Escherichia coli by deletion of bioH

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.     

Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

Web-based telemicroscopy

By taking advantage of network-based computing and the recent developments in Web interfaces, centralized research facilities housing specialized and unique imaging instruments along with associated high-performance computing can be made available to researchers for use from their own laboratories. In addition to increasing access and utilization of these facilities, operation over the Internet is expected to enhance research by facilitating collaboration between researchers. We describe the implementation of a platform-independent Web-based system written in Java that supplements automated functions with video-guided interactive, collaborative remote control and data acquisition from an intermediate-high-voltage electron microscope.     

Donor–Acceptor–Acceptor's Molecules for Vacuum‐Deposited Organic Photovoltaics with Efficiency Exceeding 9%

Three vacuum‐deposited donor–acceptor–acceptor (d–a–a') small molecule donors are studied with different side chains attached to an asymmetric heterotetracene donor block for use in high efficiency organic photovoltaics (OPVs). The donor with an isobutyl side chain yields the highest crystal packing density compared to molecules with 2‐ethylhexyl or n‐butyl chains, leading to the largest absorption coefficient and short circuit current in an OPV. It also exhibits a higher fill factor, consistent with its preferred out‐of‐plane molecular π–π stacking arrangement that facilitates charge transport in the direction perpendicular to the substrate. A power conversion efficiency of 9.3 ± 0.5% is achieved under 1 sun intensity, AM 1.5 G simulated solar illumination, which is significantly higher than 7.5 ± 0.4% of the other two molecules. These results indicate that side chain modification of d–a–a' small molecules offers an effective approach to control the crystal packing configuration, thereby improving the device performance.     

Function of a membrane-embedded domain evolutionarily multiplied in the GPI lipid anchor pathway proteins PIG-B,PIG-M,PIG-U,PIG-W,PIG-V,and PIG-Z

Distant homology relationships among proteins with many transmembrane regions (TMs) are difficult to detect as they are clouded by the TMs’ hydrophobic compositional bias and mutational divergence in connecting loops. In the case of several GPI lipid anchor biosynthesis pathway components, the hidden evolutionary signal can be revealed with dissectHMMER, a sequence similarity search tool focusing on fold-critical, high complexity sequence segments. We find that a sequence module with 10 TMs in PIG-W, described as acyl transferase, is homologous to PIG-U, a transamidase subunit without characterized molecular function, and to mannosyltransferases PIG-B, PIG-M, PIG-V and PIG-Z. We conclude that this new, membrane-embedded domain named BindGPILA functions as the unit for recognizing, binding and stabilizing the GPI lipid anchor in a modification-competent form as this appears the only functional aspect shared among all proteins. Thus, PIG-U's likely molecular function is shuttling/presenting the anchor in a productive conformation to the transamidase complex.     

Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome “maturation” requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd‐2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.     

High histone variant H3.3 content in mouse prospermatogonia suggests a role in epigenetic reformatting

Histone variants can incorporate into the nucleosome outside of S-phase. Some are known to play important roles in mammalian germ cell development, this cell lineage being characterized by long phases of quiescence, a protracted meiotic phase, and genome-wide epigenetic reformatting events. The best known example of such an event is the global-scale erasure of DNA methylation in sexually indifferent primordial germ cells, then its re-establishment in fetal prospermatogonia and growing oocytes. Histone H3 and its post-translationally modified forms provide important waypoints in the establishment of epigenetic states. Using mass spectrometry and immunoblotting, we show that the H3.3 replacement variant is present at an unusually high amount in mouse prospermatogonia at the peak stage of global DNA methylation re-establishment. We speculate that H3.3 facilitates this process through achieving a greater level of accessibility of chromatin modifiers to DNA.     

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.