GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.

BACKGROUND: The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. RESULTS: We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. CONCLUSIONS: PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of "finished grade" because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

Polydom/SVEP1 is a ligand for integrin α9ß1.

A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9ß1. However, their affinities for integrin α9ß1 are apparently much lower than those of other integrins (e.g. α3ß1, α5ß1, and α8ß1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9ß1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9ß1-dependent manner and binds directly to recombinant integrin α9ß1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636-2644) in the 21st complement control protein domain was involved in the binding to integrin α9ß1 and that Glu(2641) was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9ß1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9ß1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9ß1 that functions as a physiological ligand in vivo.

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication.

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC50=0.74-4.92 µM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC50=29.81 µM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.

Antigenic analysis of highly pathogenic avian influenza virus H5N1 sublineages co-circulating in Egypt.

Highly pathogenic avian influenza virus H5N1 has spread across Eurasia and Africa, and outbreaks are now endemic in several countries, including Indonesia, Vietnam and Egypt. Continuous circulation of H5N1 virus in Egypt, from a single infected source, has led to significant genetic diversification with phylogenetically separable sublineages, providing an opportunity to study the impact of genetic evolution on viral phenotypic variation. In this study, we analysed the phylogeny of H5 haemagglutinin (HA) genes in influenza viruses isolated in Egypt from 2006 to 2011 and investigated the effect of conserved amino acid mutations in the HA genes in each of the sublineages on their antigenicity. The analysis showed that viruses in at least four sublineages still persisted in poultry in Egypt as of 2011. Using reverse genetics to generate HA-reassortment viruses with specific HA mutations, we found antigenic drift in the HA in two influenza virus sublineages, compared with the other currently co-circulating influenza virus sublineages in Egypt. Moreover, the two sublineages with significant antigenic drift were antigenically distinguishable. Our findings suggested that phylogenetically divergent H5N1 viruses, which were not antigenically cross-reactive, were co-circulating in Egypt, indicating that there was a problem in using a single influenza virus strain as seed virus to produce influenza virus vaccine in Egypt and providing data for designing more efficacious control strategies in H5N1-endemic areas.

Metagenomic analysis of DNA viruses in a wastewater treatment plant in tropical climate.

Viruses have been detected in the different stages of wastewater treatment plants (WWTPs) at concentrations of 10(8) -10(10) ml(-1) of virus-like particles (VLPs), 10-1000 times higher than in natural aquatic environments, suggesting that WWTPs can be considered as an important reservoir and source of viruses. This study revealed novel diversity and function with the DNA viral communities in the influent, activated sludge, anaerobic digester, and effluent of a domestic WWTP using metagenomics. WWTP was a very specific environment, with less than 5% of the > 936 000 metagenomic sequences obtained (∼70-119 Mbp per sample) similar to sequences present in other environmental viromes. Many viruses found in the WWTP were novel, resulting in only < 5-20% of the reads being phylogenetically or functionally assigned. DNA metabolism was observed as the most abundant function with DNA methylase detected at levels 4.2-fold higher than other published viromes, while carbohydrate and amino acids metabolisms were 3.7- and 4.2-fold less abundant respectively. These specific aspects of the WWTP community functions are likely due to high biomass concentration, turnover rate and microbial activity in WWTPs, and likely include mechanisms that help viruses increase their infectivity. Among ∼500 genotypes estimated in individual WWTP viromes, > 82% were shared. These data suggested that VLPs of most viral types could be present between 1 and 30 days in the process before they were discharged. Viruses in WWTP and the discharged ones can have potential impacts on the functioning of the wastewater treatment system and on the dynamics of microbial community in the surrounding aquatic environments respectively.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition.

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Identification and characterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal genetic lineage and diversity of pathogenic machinery beyond the species level.

Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome sequencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealed the presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1 and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in human pathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V. parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA region of approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, was sequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SS in this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SS apparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showed that all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogenetic analysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemolyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also found among pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distinct types are distributed not only within a species but also beyond the species level and provide a new insight into the pathogenicity and evolution of Vibrio species.

Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody.

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.

Metagenomic diagnosis of bacterial infections.

To test the ability of high-throughput DNA sequencing to detect bacterial pathogens, we used it on DNA from a patient's feces during and after diarrheal illness. Sequences showing best matches for Campylobacter jejuni were detected only in the illness sample. Various bacteria may be detectable with this metagenomic approach.

Computational search for over-represented 8-mers within the 5'-regulatory regions of 634 mouse testis-specific genes.

Accumulation of microarray data has enabled the computational analysis of gene expressions in various tissues. Although the genes showing testis-specific expression are most abundant among the genes exhibiting tissue-specific expression, no systematic study has been conducted for over-represented motifs within their regulatory regions. We have identified 117 over-represented 8-mers that appeared 2648 times within the regulatory regions of 634 testis-specific genes. Of these, 64 over-represented 8-mers were significantly more frequent in the regulatory regions of testis-specific genes than in those of non-testis-specific genes. In this group of 8-mers, 4 8-mers differed from the canonical cAMP response element (CRE) 8-mer by 1 letter, but the canonical CRE was not included in this group. We consider that CRE-like 8-mers participate in the regulatory expression of testis-specific genes to a greater extent than the canonical CRE 8-mer.

In silico study of a novel gene evolved from an ancestral SVIP gene and highly expressed in the adult mouse testes.

We found that a cDNA clone isolated from a mouse testis cDNA library, 1700015G11 (Mmu_15G11), corresponded to the most highly expressed testis-specific mRNA in the adult mouse. Although the Mmu_15G11 cDNA is predicted to encode a small protein consisting of 67 amino acid residues, it has not yet been functionally annotated and has been designated as an unclassifiable clone. Since the Mmu_15G11 protein possibly has a pivotal role in spermatogenesis, we initiated an in silico study of this clone, and revealed that an ancestral gene of 15G11 genes evolved from an ancestral gene for mammalian small valosin-containing protein-interacting protein (SVIP) genes by gene duplication. Although SVIP protein reportedly participates in endoplasmic reticulum-related protein degradation, 15G11 protein is predicted to be a nuclear protein and possibly participates in the interaction between proteins and nuclear DNA.

In silico study of whey-acidic-protein domain containing oral protease inhibitors.

Since whey-acidic-protein domain (WAP) containing protease inhibitors such as SLPI (secretory leukocyte protease inhibitor) and elafin (elastase-specific inhibitor) have antimicrobial activities and are thought to play critical roles in mucosal defenses, we are interested in these protease inhibitors. By accessing the Novartis mouse expression database, we found that the four WAP family members, SLPI, WFDC2, WFDC5, and WFDC12, are highly expressed in the oral organs, such as the trachea, tongue, and salivary glands. Since their WAP domains play pivotal roles in the antimicrobial and/or antiprotease activities and their application in therapeutics are expected to have practical value, we collected 98 WAP homologues and tried to predict their physiological functions by analyzing their amino acid sequence structures. From the multiple alignments of amino acid sequences, we predicted that most of the mammalian N-terminal WAP domains derived from SLPIs and the WAP domains derived from WFDC12s have antimicrobial activities, whereas most of the mammalian C-terminal WAP domains derived from SLPIs and the WAP domains derived from elafins have antiprotease activities. From the phylogenetic tree, it was revealed that an ancestral WAP protein initially diverged into the WFDC5-C WAP domain and the ancestral protein for the other WAP domains. Subsequently, the ancestral protein for the other WAP domains diverged into two ancestral proteins, one for elafin and SLPI-C WAP domains and the other, for SLPI-N, WFDC15b, WFDC12, and WFDC5-N WAP domains, respectively. Moreover, the tree indicated that the WFDC5-N and WFDC12 WAP domains share a common ancestral protein.

Analysis of invariant sequences in 266 complete genomes.

To date, the complete genome sequences of more than 250 organisms have been determined. This information can now be used to determine whether there exist any invariant sequences that are conserved among all organisms, from bacteria to plants, animals, and humans. The existence of invariant sequences would strongly suggest that these sequences have been inherited unchanged from the last common ancestor of all life, and that they have essential functions. We have developed a new software program to identify invariant sequences conserved among the currently sequenced genomes and applied this analysis to the complete genome sequences of 266 organisms. We have identified 3 invariant DNA sequences longer than or equal to 11 bp and 6 invariant amino acid sequences longer than or equal to 6 aa. The longest invariant DNA sequence, AAGTCGTACAAGGT (15 bp), was found in the 16S/18S rRNA gene. Two 8 aa sequences, GHVDHGKT in IF2 and EF-Tu and DTPGHVDF in EF-G, were the longest invariant amino acid sequences detected. These sequences could be essential elements from the genome of the last common ancestor and may have remained unchanged throughout evolution.

Fungal ITS1 Deep-Sequencing Strategies to Reconstruct the Composition of a 26-Species Community and Evaluation of the Gut Mycobiota of Healthy Japanese Individuals.

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio's circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells.

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.

Genetic analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: Gene expression profiles in the kidneys.

Spontaneously hypertensive rats (SHRs) and stroke-prone SHRs (SHRSP) are frequently used as models not only of essential hypertension and stroke, but also of attention-deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto (WKY) rats are normally used as controls in these studies. In the present study, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD using these rats. We previously analyzed gene expression profiles in the adrenal glands and brain. Since the kidneys can directly influence the functions of the cardiovascular, endocrine and sympathetic nervous systems, gene expression profiles in the kidneys of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks old, a period in which rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between the SHRs and WKY rats and also between the SHRSP and SHRs. A total of 232 unique genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated as SHR- and SHRSP-specific genes. Candidate genes were then selected using two different web tools: the 1st tool was the Database for Annotation, Visualization and Integrated Discovery (DAVID), which was used to search for significantly enriched genes and categorized them using Gene Ontology (GO) terms, and the 2nd was Ingenuity Pathway Analysis (IPA), which was used to search for interactions among SHR- and also SHRSP­specific genes. The analyses of SHR-specific genes using IPA revealed that B-cell CLL/lymphoma 6 (Bcl6) and SRY (sex determining region Y)-box 2 (Sox2) were possible candidate genes responsible for causing hypertension in SHRs. Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), angiotensin II receptor-associated protein (Agtrap) and apolipoprotein H (Apoh) were possible candidate genes responsible for triggering strokes. Since our results revealed that SHRSP-specific genes isolated from the kidneys of rats at 6 weeks of age, included 6 genes related to Huntington's disease, we discussed the genetic association between ADHD and Huntington's disease.

Study of transcripts from AC010088, a 199,485 bp fragment of the human Y chromosome located in the azoospermia factor region c.

Deletions on the long arm of the human Y chromosome are associated with male infertility. In this work, we studied transcripts of a 199,485 bp long fragment of the Yq11 region (GenBank accession number, AC010088) located in the AZFc (azoospermia factor region c), and characterized their gene structures. After masking repetitive elements, we searched human mRNA Refseqs (reference sequences), a dbEST (database of expressed sequence tags) and a non-redundant nucleic acid database for the mRNAs and ESTs corresponding to the AC010088 using the BLAST programs at the NCBI (National Center for Biotechnology Information) site. Our findings are summarized as follows: i) BPY2 (testis basic protein on Y, 2), DAZ1 (deleted in azoospermia 1), TTY4 (testis transcript Y 4) mRNAs and 23 ESTs were found; ii) Eighteen of 23 ESTs were transcripts of the DAZ gene(s), one EST was a transcript of TTY4 gene, and the remaining 4 probably corresponded to 4 different pseudogenes; iii) DAZ gene(s) were expressed not only in testis, but also in lung carcinoma cells, stomach and Ewing's sarcoma cells; iv) beta-satellite clusters were present around and within the BPY2 and TTY4 gene region; v) In this study, TTY4, BPY2 and DAZ1 genes were mapped precisely to the AC010088 region.

Analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: gene expression profiles in the brain.

Spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) are frequently used as rat models not only of essential hypertension and stroke, but also of attention-deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto rats (WKY) are used as the control rats in these cases. An increasing number of studies has demonstrated the critical role of the central nervous system in the development and maintenance of hypertension. In a previous study, we analyzed the gene expression profiles in the adrenal glands of SHR. Thus, in this study, we analyzed gene expression profiles in the brains of SHR in order to identify the genes responsible for causing hypertension and stroke, as well as those involved in ADHD. Using genome-wide microarray technology, we examined the gene expression profiles in the brains of 3 rat strains (SHR, SHRSP and WKY) when the rats were 3 and 6 weeks of age, a period in which the rats are considered to be in a pre-hypertensive state. Gene expression profiles in the brain were compared between SHR and WKY, and between SHRSP and SHR. A total of 179 genes showing a >4- or <-4-fold change in expression were isolated, and candidate genes were selected using two different web tools: the first tool was the Database for Annotation, Visualization and Integrated Discovery (DAVID), which was used to search for significantly enriched genes, and categorized them using Gene Ontology (GO) terms, and the second was the network explorer of Ingenuity Pathway Analysis (IPA), which was used to search for interaction networks among SHR- and SHRSP-specific genes. The IPA of SHR-specific genes revealed that prostaglandin E receptor 4 (Ptger4) is one of the candidate genes responsible for causing hypertension in SHR, and that albumin (Alb) and chymase 1 (Cma1) are also responsible for causing hypertension in SHR in the presence of angiotensinogen (Agt). Similar analyses of SHRSP-specific genes revealed that the angiotensin II receptor-associated gene (Agtrap) interacts with the FBJ osteosarcoma oncogene (Fos), and with the angiotensin II receptor type-1b (Agtr1b). As Agtrap and Agtr1b not only participate in the 'uptake of norepinephrine' and 'blood pressure', but also in the 'behavior' of SHRSP at 6 weeks of age, our data demonstrate a close association between hypertension and ADHD.

Generation of rodent malaria parasites with a high mutation rate by destructing proofreading activity of DNA polymerase δ.

Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.