Trimethylamine N-oxide (TMAO), which is oxidized from trimethylamine (TMA) by hepatic flavin-containing monooxygenases (FMOs), promotes the development of atherosclerosis and is a new target for the prevention and treatment of cardiovascular disease from the perspective of intestinal flora. TMA is transformed by intestinal flora from TMA-containing nutrients, such as choline. Some small molecular agents lower serum TMAO and/or cecal TMA levels. However, probiotics that can effectively reduce serum TMAO levels are currently lacking. In this work, five potentially probiotic strains were administered to mice supplemented with 1.3% choline. Only Lactobacillus plantarum ZDY04 significantly reduced serum TMAO and cecal TMA levels by modulating the relative abundance of the families Lachnospiraceae, Erysipelotrichaceae and Bacteroidaceae and the genus Mucispirillum in mice and not by influencing the expression levels of hepatic FMO3 and metabolizing choline, TMA, and TMAO. In addition, L. plantarum ZDY04 can significantly inhibit the development of TMAO-induced atherosclerosis in ApoE-/- 1.3% choline-fed mice as compared with the untreated PBS group. In conclusion, the use of L. plantarum ZDY04 may be an alternative approach to reduce serum TMAO levels and TMAO-induced atherosclerosis in mice.

BackgroundThe impact of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV.Methodology/Principal FindingsBy comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII.Conclusions/SignificanceUsing RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide.

d ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin-cry1-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin-Cry1-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ϳ65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.