L-Ascorbate Biosynthesis Involves Carbon Skeleton Rearrangement in the Nematode Caenorhabditis elegans

Ascorbate (AsA) is required as a cofactor and is widely distributed in plants and animals. Recently, it has been suggested that the nematode Caenorhabditis elegans also synthesizes AsA. However, its biosynthetic pathway is still unknown. To further understand AsA biosynthesis in C. elegans, we analyzed the incorporation of the 13C atom into AsA using gas chromatography-mass spectrometry (GC-MS) in worms fed with D-Glc (1-13C)-labeled Escherichia coli. GC-MS analysis revealed that AsA biosynthesis in C. elegans, similarly to that in mammalian systems, involves carbon skeleton rearrangement. The addition of L-gulono-1,4-lactone, an AsA precursor in the mammalian pathway, significantly increased AsA level in C. elegans, whereas the addition of L-galactono-1,4-lactone, an AsA precursor in the plant and Euglena pathway, did not affect AsA level. The suppression of E03H4.3 (an ortholog of gluconolactonase) or the deficiency of F54D5.12 (an ortholog of L-gulono-1,4-lactone oxidase) significantly decreased AsA level in C. elegans. Although N2- and AsA-deficient F54D5.12 knockout mutant worm (tm6671) morphologies and the ratio of collagen to non-collagen protein did not show any significant differences, the mutant worms exhibited increased malondialdehyde levels and reduced lifespan compared with the N2 worms. In conclusion, our findings indicate that the AsA biosynthetic pathway is similar in C. elegans and mammals

Long-term tolerability and effectiveness of raltegravir in Japanese patients: Results from post-marketing surveillance.

Antiretroviral agents are approved in Japan based on non-clinical and clinical data reported from overseas. Neither the long-term tolerability nor the effectiveness of raltegravir or other integrase strand transfer inhibitors in Japan is known. This study reports on the long-term tolerability and effectiveness of raltegravir in Japanese clinical practice using data collected through approximately 9 years of post-marketing surveillance. This observational survey used data on human immunodeficiency virus (HIV) infected patients initiated treatment with raltegravir between 2008 and 2017 in the HIV-related drug (HRD) cooperative survey to assess the safety and effectiveness of raltegravir in real world clinical practice. There were totally 1,303 patients prescribed raltegravir across 30 institutions; 1,293 patients and 1,178 patients were included for the safety and effectiveness analyses, respectively. The overall risk of adverse drug reaction was 17.25%, with abnormal hepatic function and hyperlipidaemia (500 cells/μL in treatment-naïve and treatment-experienced patients after 3 years and 4 years of treatment, respectively. In Japanese HIV patients, long-term treatment with raltegravir is well-tolerated and effective at viral suppression as measured by HIV-1 RNA levels and subsequent change in CD4+ cell counts. Such benefits can be expected for not only treatment-naïve but also treatment-experienced patients

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential

L-Ascorbate Biosynthesis Involves Carbon Skeleton Rearrangement in the Nematode Caenorhabditis elegans

Ascorbate (AsA) is required as a cofactor and is widely distributed in plants and animals. Recently, it has been suggested that the nematode Caenorhabditis elegans also synthesizes AsA. However, its biosynthetic pathway is still unknown. To further understand AsA biosynthesis in C. elegans, we analyzed the incorporation of the 13C atom into AsA using gas chromatography-mass spectrometry (GC-MS) in worms fed with D-Glc (1-13C)-labeled Escherichia coli. GC-MS analysis revealed that AsA biosynthesis in C. elegans, similarly to that in mammalian systems, involves carbon skeleton rearrangement. The addition of L-gulono-1,4-lactone, an AsA precursor in the mammalian pathway, significantly increased AsA level in C. elegans, whereas the addition of L-galactono-1,4-lactone, an AsA precursor in the plant and Euglena pathway, did not affect AsA level. The suppression of E03H4.3 (an ortholog of gluconolactonase) or the deficiency of F54D5.12 (an ortholog of L-gulono-1,4-lactone oxidase) significantly decreased AsA level in C. elegans. Although N2- and AsA-deficient F54D5.12 knockout mutant worm (tm6671) morphologies and the ratio of collagen to non-collagen protein did not show any significant differences, the mutant worms exhibited increased malondialdehyde levels and reduced lifespan compared with the N2 worms. In conclusion, our findings indicate that the AsA biosynthetic pathway is similar in C. elegans and mammals