Keisuke Ueno

Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro

The effects of chitin and its derivatives on the proliferation of fibroblasts and on the production of cytokines were examined in vitro. Chitin and its derivatives showed almost no acceleratory effect on the proliferation of cultured fibroblasts. On the contrary, high-concentration 500 micrograms ml-1) D-glucosamine cultures supplemented with or without a 10% fetal calf serum (FCS) supplementation showed a significant (P < 0.05) reduction in the rate of proliferation of L929 fibroblast cells relative to control. High-concentration chitosan cultures supplemented with 10% FCS showed a significant (P < 0.05) reduction in the rate of L929 fibroblast proliferation. However, the inhibition of cell proliferation by high concentrations of chitosan did not show in cultures without FCS. Interleukin-8 (IL-8) was induced in the supernatants of rat primary cultured dermal fibroblasts stimulated with chitin and its derivatives. Chitin and its derivatives did not stimulate the production of IL-6 by mouse dermal primary cultured fibroblasts. IL-1 alpha, IL-1 beta and tumour necrosis factor-alpha were not detected in the fibroblast supernatants. These observations support the notion that cell proliferation is accelerated indirectly by chitin and its derivatives when these materials are used in vivo. In vivo findings of a angiogenesis and migration of neutrophils may be due to persistent release of IL-8 from fibroblasts.

Biosynthesis of hetero-polysaccharides by Acetobacter xylinum - Synthesis and characterization of metal-ion adsorptive properties of partially carboxymethylated cellulose

Abstract Biological reconstruction of water-soluble carboxymethylated cellulose (CMC; D.S. =0.47) has been achieved by culturing Acetobacter xylinum in medium containing CMC and d -glucose to give a novel hetero-polysaccharide having a carboxymethyl function. The novel extracellular polysaccharide, carboxymethylated-bacterial cellulose (CM-BC), had an ion exchange ability with enhanced specific adsorption for lead and uranyl ions compared to the original CMC and bacterial cellulose. The contribution of the hydroxy group at C-2 was confirmed by applying carboxymethylated chitin, which possesses acetamido group at C-2 of the glucose residue, as the carbon source of the incubation.

CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.

Metagenomic analysis of the shrew enteric virome reveals novel viruses related to human stool-associated viruses.

Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.

A nairovirus isolated from African bats causes haemorrhagic gastroenteritis and severe hepatic disease in mice

Bats can carry important zoonotic pathogens. Here we use a combination of next-generation sequencing and classical virus isolation methods to identify novel nairoviruses from bats captured from a cave in Zambia. This nairovirus infection is highly prevalent among giant leaf-nosed bats, Hipposideros gigas (detected in samples from 16 individuals out of 38). Whole-genome analysis of three viral isolates (11SB17, 11SB19 and 11SB23) reveals a typical bunyavirus tri-segmented genome. The strains form a single phylogenetic clade that is divergent from other known nairoviruses, and are hereafter designated as Leopards Hill virus (LPHV). When i.p. injected into mice, the 11SB17 strain causes only slight body weight loss, whereas 11SB23 produces acute and lethal disease closely resembling that observed with Crimean–Congo Haemorrhagic Fever virus in humans. We believe that our LPHV mouse model will be useful for research on the pathogenesis of nairoviral haemorrhagic disease.

Proteomic profiling reveals compartment‐specific, novel functions of ascidian sperm proteins

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X‐100‐soluble and ‐insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS–PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non‐redundant proteins were identified by mass spectrometry (MALDI‐TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm‐specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca2+‐binding proteins in axonemes, localization of testis‐specific serine/threonine kinase, and the presence of G‐protein coupled signaling and ubiquitin pathway in sperm flagella. Mol. Reprod. Dev. 78:529–549, 2011.

Distinct Lineages of Bufavirus in Wild Shrews and Nonhuman Primates

Viral metagenomic analysis identified a new parvovirus genome in the intestinal contents of wild shrews in Zambia. Related viruses were detected in spleen tissues from wild shrews and nonhuman primates. Phylogenetic analyses showed that these viruses are related to human bufaviruses, highlighting the presence and genetic diversity of bufaviruses in wildlife.

Resampling nucleotide sequences with closest-neighbor trimming and its comparison to other methods.

A large number of nucleotide sequences of various pathogens are available in public databases. The growth of the datasets has resulted in an enormous increase in computational costs. Moreover, due to differences in surveillance activities, the number of sequences found in databases varies from one country to another and from year to year. Therefore, it is important to study resampling methods to reduce the sampling bias. A novel algorithm–called the closest-neighbor trimming method–that resamples a given number of sequences from a large nucleotide sequence dataset was proposed. The performance of the proposed algorithm was compared with other algorithms by using the nucleotide sequences of human H3N2 influenza viruses. We compared the closest-neighbor trimming method with the naive hierarchical clustering algorithm and -medoids clustering algorithm. Genetic information accumulated in public databases contains sampling bias. The closest-neighbor trimming method can thin out densely sampled sequences from a given dataset. Since nucleotide sequences are among the most widely used materials for life sciences, we anticipate that our algorithm to various datasets will result in reducing sampling bias.

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

ELM: enhanced lowest common ancestor based method for detecting a pathogenic virus from a large sequence dataset

BackgroundEmerging viral diseases, most of which are caused by the transmission of viruses from animals to humans, pose a threat to public health. Discovering pathogenic viruses through surveillance is the key to preparedness for this potential threat. Next generation sequencing (NGS) helps us to identify viruses without the design of a specific PCR primer. The major task in NGS data analysis is taxonomic identification for vast numbers of sequences. However, taxonomic identification via a BLAST search against all the known sequences is a computational bottleneck.DescriptionHere we propose an enhanced lowest-common-ancestor based method (ELM) to effectively identify viruses from massive sequence data. To reduce the computational cost, ELM uses a customized database composed only of viral sequences for the BLAST search. At the same time, ELM adopts a novel criterion to suppress the rise in false positive assignments caused by the small database. As a result, identification by ELM is more than 1,000 times faster than the conventional methods without loss of accuracy.ConclusionsWe anticipate that ELM will contribute to direct diagnosis of viral infections. The web server and the customized viral database are freely available at http://bioinformatics.czc.hokudai.ac.jp/ELM/.