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Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.

b Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6؋His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P araS-SD , an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P araS-SD directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6؋His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6؋His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6؋His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site. C urrently, studying functions of thermophilic archaeal proteins, such as the enzymes involved in DNA replication, gene transcription, and protein translation (10), as well as clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) proteins (11, 32), relies almost exclusively on production of recombinant protein from a mesophilic bacterial host such as Escherichia coli and characterization of purified enzymes. However, there is a major drawback in this approach: many thermophilic proteins are insoluble in mesophilic bacterial cells, forming inclusion bodies. Apparently, producing recombinant thermophilic proteins in a homologous host should resolve the problem of protein solubility.For the past few decades, consistent efforts have been devoted to developing protein expression systems for hyperthermophilic organisms and to establishing genetic tools for studying functions of archaeal genes. As a result, methodologies for gene inactivation and genetic complementation have been established for several model archaeal organisms (reviewed in reference 15), including three hyperthermophilic archaea, the euryarchaea Thermococcus kodakarensis (22) and Pyrococcus furiosus (30), and the crenarchaeon Sulfolobus. While both T. kodakarensis and P. furiosus require an anaerobic condition for growth, Sulfolobus species grow aerobically, which is advantageous for laboratory manipulation. Currently, three Sulfolobus species have be...

SummaryCaenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed 'WormFarm', an integrated microfluidic device for culturing nematodes. Cohorts of 30-50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof-of-principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate-based assay for both RNAi-mediated and dietary-induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age-associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo-3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.

Self-switchable polymerization is an attractive strategy for precisely controlling the microstructures and monomer sequences of polymers. To date, catalysts for the polymerization are generally limited to metal complex catalysts and some organocatalysts. In this article, we report that simple, inexpensive, and environmentally benign alkali metal carboxylate catalysts smartly switch between the ring-opening alternating copolymerization of epoxides with cyclic anhydrides and the ring-opening polymerization of cyclic esters to create a single synthetic step and thus achieve sequence-controlled multiblock polyesters. This polymerization system shows extremely high effectiveness and versatility for different combinations of epoxides, cyclic anhydrides, cyclic esters, and initiators. As a result, various types of complex block copolymers, such as AB diblocks, BAB triblocks, star copolymers, hyperbranched copolymers, and CAB triblocks, can be simply prepared and postpolymerization modification can be performed. As a proof of concept, polyester-based elastomers and adhesives were successfully synthesized via one-step procedures by reasonably designing the monomer structures of triblock copolymers, showing great potential for industrial applications of polyesters.

The innovative transfer printing fabrication procedure is facile and inexpensive, and thus applied in various wearable displays.

Using a case-control method, we studied the role of environmental risk factors and viral infection in the development of Parkinson''s disease (PD) in China. Ninety-three PD patients and 186 controls were investigated with a questionnaire and from most of them, blood was taken to test the antibody levels of four virus (measles, rubella, HSV-1, CMV) IgG. The study result showed that positive family history, living near rubber plants, drinking river-water were associated with an increased risk of developing PD. In contrast, living in small cities, drinking well-water, drinking hard-liquor frequently, were associated with a decreased risk for PD. PD cases and controls did not differ with respect to other factors investigated including smoking and viral infection. These findings suggest that some environmental factors may be related to the development of PD, but further standardized studies will be required to confirm our results.

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