Whole genome sequencing of Entamoeba nuttalli reveals mammalian host-related molecular signatures and a novel octapeptide-repeat surface protein.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection.

Loss of Bacitracin Resistance Due to a Large Genomic Deletion among Bacillus anthracis Strains.

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

Population Structure and Local Adaptation of MAC Lung Disease Agent Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.

Filling in the Gap of Human Chromosome 4: Single Molecule Real Time Sequencing of Macrosatellite Repeats in the Facioscapulohumeral Muscular Dystrophy Locus.

A majority of facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of macrosatellite repeats called D4Z4 that are located in the subtelomeric region of human chromosome 4q35. Sequencing the FSHD locus has been technically challenging due to its long size and nearly identical nature of repeat elements. Here we report sequencing and partial assembly of a BAC clone carrying an entire FSHD locus by a single molecule real time (SMRT) sequencing technology which could produce long reads up to about 18 kb containing D4Z4 repeats. De novo assembly by Hierarchical Genome Assembly Process 1 (HGAP.1) yielded a contig of 41 kb containing all but a part of the most distal D4Z4 element. The validity of the sequence model was confirmed by an independent approach employing anchored multiple sequence alignment by Kalign using reads containing unique flanking sequences. Our data will provide a basis for further optimization of sequencing and assembly conditions of D4Z4.

Amelioration of angiotensin II-induced salt-sensitive hypertension by liver-type fatty acid-binding protein in proximal tubules.

Inappropriate activation of the intrarenal renin-angiotensin system induces generation of reactive oxygen species and tubulointerstitial inflammation, which contribute to salt-sensitive hypertension (SSHT). Liver-type fatty acid-binding protein is expressed in proximal tubules in humans, but not in rodents, and may play an endogenous antioxidative role. The objective of the present study was to examine the antioxidative effect of liver-type fatty acid-binding protein on post-angiotensin II SSHT model in transgenic mice with selective overexpression of human liver-type fatty acid-binding protein in the proximal tubules. The transgenic mice showed marked protection against angiotensin II-induced SSHT. Overexpression of tubular liver-type fatty acid-binding protein prevented intrarenal T-cell infiltration and also reduced reactive oxygen species generation, intrarenal renin-angiotensin system activation, and monocyte chemotactic protein-1 expression. We also performed an in vitro study using the murine proximal tubular cell lines with or without recombinant liver-type fatty acid-binding protein and murine proximal tubular cell lines transfected with human liver-type fatty acid-binding protein, and found that gene transfection of liver-type fatty acid-binding protein and, in part, recombinant liver-type fatty acid-binding protein administration had significantly attenuated angiotensin II-induced reactive oxygen species generation and the expression of angiotensinogen and monocyte chemotactic protein-1 in murine proximal tubular cell lines. These findings indicated that liver-type fatty acid-binding protein in the proximal tubules may protect against angiotensin II-induced SSHT by attenuating activation of the intrarenal renin-angiotensin system and reducing oxidative stress and tubulointerstitial inflammation. Present data suggest that liver-type fatty acid-binding protein in the proximal tubules may be a novel therapeutic target for SSHT.

Protective effects of L-type fatty acid-binding protein (L-FABP) in proximal tubular cells against glomerular injury in anti-GBM antibody-mediated glomerulonephritis.

BACKGROUND: In glomerulonephritis (GN), an overload of free fatty acids (FFA) bound to albumin in urinary protein may induce oxidative stress in the proximal tubules. Human liver-type fatty acid-binding protein (hL-FABP) expressed in human proximal tubules, but not rodents, participates in intracellular FFA metabolism and exerts anti-oxidative effects on the progression of tubulointerstitial damage. We examined whether tubular enhancement of this anti-oxidative action modulates the progression of glomerular damage in immune-mediated GN in hL-FABP chromosomal gene transgenic (Tg) mice. METHODS: Anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) was induced in Tg and wild-type mice (WT). Proteinuria, histopathology, polymorphonuclear (PMN) influx, expression of tubulointerstitial markers for oxidative stress 4-hydroxy-2-Nonenal (HNE) and fibrosis (α-smooth muscle actin), proximal tubular damage (Kim-1), Peroxisome Proliferator-Activated Receptor γ (PPAR γ) and inflammatory cytokines [Monocyte Chemotactic Protein-1, tumor necrosis factor-alpha (TNF-α) and Transforming growth factor beta (TGF-ß)] were analyzed. The mice were also treated with an angiotensin type II receptor blocker (ARB). RESULTS: The urinary protein level in Tg mice decreased significantly during the acute phase (~Day 5). Tg mice survived for a significantly longer time than WT mice, with an attenuation of tubulointerstitial damage score and expression of each tubulointerstitial damage marker observed at Day 7. Expression of inflammatory cytokines on Day 7 was higher in WT mice than Tg mice and correlated strongly with PPARγ expression in WT mice, but not in Tg mice. Interestingly, Tg mice showed insufficient PMN influx at 3 and 6 h, with simultaneous elevation of urinary L-FABP and reduction in HNE expression. The two strains of mice showed different types of glomerular damage, with mild mesangial proliferation in Tg mice and severe endothelial swelling with vascular thrombosis in WT mice. The glomerular damage in Tg mice was improved by administration of an ARB. CONCLUSIONS: The present experimental model suggests that tubular enhancement of L-FABP may protect mice with anti-GBM GN from progression of both tubulointerstitial and glomerular injury.

Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

BACKGROUND: Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. CONCLUSIONS/SIGNIFICANCE: Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

Three cases of PR3-ANCA positive subacute endocarditis caused by attenuated bacteria (Propionibacterium, Gemella, and Bartonella) complicated with kidney injury.

Subacute bacterial endocarditis (SBE) associated with antiproteinase-3 antineutrophil cytoplasmic antibodies (PR3-ANCA) has previously been reported in 10 cases of Streptococcus viridans and in 1 case of Escherichia faecalis infection. Most of these patients had hypocomplementemia and were positive for several autoantibodies. The infections in most of these patients showed good responses to antibiotic treatment. We report three patients with ANCA-positive SBE, which was induced by attenuated slow-growing intracellular pathogens; these patients had severe complications, such as acute kidney injury, cerebral embolism, and aortic valve destruction.

Protective effects of tubular liver-type fatty acid-binding protein against glomerular damage in murine IgA nephropathy.

BACKGROUND: Liver-type fatty acid-binding protein (L-FABP) in proximal tubules was reported to have renoprotective roles in experimental tubulointerstitial diseases via its anti-oxidative properties. Since tubuloglomerular cross-talk was recently discussed in the progression of renal diseases, to investigate whether tubular L-FABP may have an impact on the progression of glomerular damage, we induced IgA nephropathy (IgAN) in mice (Tg) transgenically tubular overexpressing human L-FABP (hL-FABP). METHODS: We reconstituted IgAN by bone marrow transplantation (BMT) from IgAN-prone mice into Tg and wild-type (WT) mice. Renal damage was evaluated at 6 and 12 weeks after BMT. During in vitro experiments, mesangial cells (MC) were stimulated by aggragated IgA (AIgA), and their supernatants (AIgA-MC medium) were collected. Stable cell line of mouse proximal tubular cell (mProx) transfected with or without hL-FABP gene was cultured with the AIgA-MC medium. RESULTS: Although mesangial IgA deposition and serum IgA level were not different between WT (WT/ddY) and Tg (Tg/ddY) recipients, WT/ddY mice showed a significantly higher urinary albumin level and mesangial matrix expansion with a significantly higher glomerular damage score. Furthermore, CD68 + macrophage infiltration was also significantly attenuated in Tg/ddY mice. Up-regulation of renal hL-FABP was associated with significant suppression of renal heme oxygenase-1 (HO-1) expression and accumulation of 4-hydroxy-2-nonenal (4-HNE) and MCP-1 expression in Tg/ddY mice. In vitro experiments showed that AIgA-MC medium and recombinant TNF-α significantly up-regulated hL-FABP expression, which was partially blocked by anti-TNF-α antibody, and major mediators of oxidative stress (HO-1 and 4-HNE) and inflammation (MCP-1). Importantly, such up-regulation of the mediators in mProx with hL-FABP was significantly suppressed much more than that in mProx. CONCLUSIONS: Tubular L-FABP activated by MC-origin humoral factors may lessen progression of glomerular damage at early stages of IgAN by reducing oxidative stress and inflammatory mediators.

Pharmacogenomics of cardiovascular pharmacology: development of an informatics system for analysis of DNA microarray data with a focus on lipid metabolism.

Genome-wide gene-expression data from DNA-microarray technology and molecular-network data from computational text-mining have led to a paradigm shift in biological research. However, interpretation of the huge amount of data is a bottleneck. We have developed an informatics system, which we refer to as bioSpace Explorer, that can extract pathways and molecules of interest from genome-wide data and show the mutual relationships among these pathways and molecules. Differentiation of 3T3-L1 cells into adipocytes and the action of a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist or alpha-linolenic acid on this process was analyzed with bioSpace Explorer. The results suggested a biological basis for adipocyte differentiation and a strategy to enhance lipid oxidation in adipocytes. Clustered changes of molecules were apparent in the insulin, Wnt, and PPARgamma signaling pathways and in the lipogenesis, lipid oxidation, and lipid transport pathways during cell differentiation. A PPARgamma agonist enhanced lipid oxidation in adipocytes and alpha-linolenic acid gave similar results to the PPARgamma agonist. An analysis of sex hormone and thyroid hormone, in addition to PPARgamma signaling, suggested that these molecules are important for enhancement of lipid oxidation in adipocytes. The results indicate the utility of bioSpace Explorer for biological research on genome-wide molecular networks.

Degradation of ethyl docosahexaenoate by gamma-ray irradiation and suppression of this degradation by antioxidants.

Ethyl 4,7,10,13,16,19-docosahexaenoate (DHA-Et) dissolved in an ethanol solution or embedded in liposomes was degraded by irradiating with gamma rays in a dose-dependent manner. The degradation rate of DHA-Et embedded in liposomes was higher than that of DHA-Et dissolved in ethanol. Antioxidants suppressed the degradation of DHA-Et embedded in liposomes, the order of activity of the antioxidants being luteolin>fisetin>kaempferol>quercetin>rutin. These results suggest that the hydrophobicity (logP) of an antioxidant is one of determinants for antioxidative activity, but that a vicinal diol structure in the B ring is not favorable for the antioxidative activity.

Reduction in photostability by the esterification of beta-cryptoxanthin.

Liposomes, in which beta-carotene, beta-cryptoxanthin, zeaxanthin, beta-cryptoxanthin palmitate or beta-cryptoxanthin acetate had been embedded, were irradiated by UVA, and the rate of degradation of each carotenoid was measured. There was no significant difference in the degradation rate between beta-carotene, beta-cryptoxanthin and zeaxanthin. The degradation rates of beta-cryptoxanthin palmitate and beta-cryptoxanthin acetate were faster than that of beta-cryptoxanthin, and the degradation rate of beta-cryptoxanthin palmitate was faster than that of beta-cryptoxanthin acetate.