Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency

TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity

While hyperosmolality of the kidney medulla is essential for urinary concentration, it imposes a great deal of stress. Cells in the renal medulla adapt to the stress of hypertonicity (hyperosmotic salt) by accumulating organic osmolytes. Tonicity-responsive enhancer (TonE) binding protein (TonEBP) (or NFAT5) stimulates transcription of transporters and a synthetic enzyme for the cellular accumulation of organic osmolytes. We found that dominant-negative TonEBP reduced expression of HSP70 as well as the transporters and enzyme. Near the major histocompatibility complex class III locus, there are three HSP70 genes named HSP70-1, HSP70-2, and HSC70t. While HSP70-1 and HSP70-2 were heat inducible, only HSP70-2 was induced by hypertonicity. In the 5' flanking region of the HSP70-2 gene, there are three sites for TonEBP binding. In cells transfected with a reporter plasmid containing this region, expression of luciferase was markedly stimulated in response to hypertonicity. Coexpression of the dominant-negative TonEBP reduced the luciferase expression. Mutating all three sites in the reporter plasmid led to a complete loss of induction by hypertonicity. Thus, TonEBP rather than heat shock factor stimulates transcription of the HSP70-2 gene in response to hypertonicity. We conclude that TonEBP is a master regulator of the renal medulla for cellular protection against high osmolality via organic osmolytes and molecular chaperones.open12

RNA-seq transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis

Bovine tuberculosis, caused by infection with Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including gene expression microarrays and RNA-sequencing (RNA-seq), has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analyzed the peripheral blood leukocyte (PBL) transcriptome of eight natural M. bovis-infected and eight age- and sex-matched non-infected control Holstein-Friesian animals using RNA-seq. In addition, we compared gene expression profiles generated using RNA-seq with those previously generated using the high-density Affymetrix(®) GeneChip(®) Bovine Genome Array platform from the same PBL-extracted RNA. A total of 3,250 differentially expressed (DE) annotated genes were detected in the M. bovis-infected samples relative to the controls (adjusted P-value ≤0.05), with the number of genes displaying decreased relative expression (1,671) exceeding those with increased relative expression (1,579). Ingenuity(®) Systems Pathway Analysis (IPA) of all DE genes revealed enrichment for genes with immune function. Notably, transcriptional suppression was observed among several of the top-ranking canonical pathways including Leukocyte Extravasation Signaling. Comparative platform analysis demonstrated that RNA-seq detected a larger number of annotated DE genes (3,250) relative to the microarray (1,398), of which 917 genes were common to both technologies and displayed the same direction of expression. Finally, we show that RNA-seq had an increased dynamic range compared to the microarray for estimating differential gene expression

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA

Toxicity assessment of modified Cry1Ac1 proteins and genetically modified insect-resistant Agb0101 rice

Insect-resistant Agb0101 rice was recently developed by modifying the cry1ac1 gene by changing codon usage changes relative to the native truncated cry1ac1 gene. To assess the toxicity of genetically modified Agb0101 rice, we conducted bioinfomational comparisons of the amino acid sequences that are not similar to known toxic proteins. Sufficient quantities of mCry1Ac1 protein were produced in Escherichia coli for in vitro evaluation and animal study. We compared the amino acid sequences and molecular mass. There have the same amino acid sequences and molecular masses after purifying the modified Cry1Ac1 (mCry1Ac1) protein from highly expressed bacteria and genetically modified rice were identical. We also investigated the acute and 90-days oral toxicities. No adverse effects were observed in mice following acute oral exposure to 2,000 mg/ kg body weight mCry1Ac1 protein of body weight and 90 days oral exposure to Agb0101. These results indicate that mCry1Ac1 proteins and Agb0101 rice demonstrate no adverse effects in these tests when applied via gavage and feed, respectively.Key words: Modified Cry1Ac1, food safety assessment, toxicity, insect- resistant rice Agb0101

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice

<div><p>Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.</p></div

Ab initio study of magnetism at the TiO2/LaAlO3 interface

In this paper we study the possible relation between the electronic and magnetic structure of the TiO2/LaAlO3 interface and the unexpected magnetism found in undoped TiO2 films grown on LaAlO$_3$. We concentrate on the role played by structural relaxation and interfacial oxygen vacancies. LaAlO3 has a layered structure along the (001) direction with alternating LaO and AlO2 planes, with nominal charges of +1 and -1, respectively. As a consequence of that, an oxygen deficient TiO2 film with anatase structure will grow preferently on the AlO2 surface layer. We have therefore performed ab-initio calculations for superlattices with TiO2/AlO2 interfaces with interfacial oxygen vacancies. Our main results are that vacancies lead to a change in the valence state of neighbour Ti atoms but not necessarily to a magnetic solution and that the appearance of magnetism depends also on structural details, such as second neighbor positions. These results are obtained using both the LSDA and LSDA+U approximations.Comment: Accepted for publication in Journal of Materials Scienc

Six-dimensional (1,0) effective action of F-theory via M-theory on Calabi-Yau threefolds

The six-dimensional effective action of F-theory compactified on a singular elliptically fibred Calabi-Yau threefold is determined by using an M-theory lift. The low-energy data are derived by comparing a circle reduction of a general six-dimensional (1,0) gauged supergravity theory with the effective action of M-theory on the resolved Calabi-Yau threefold. The derivation includes six-dimensional tensor multiplets for which the (anti-) self-duality constraints are imposed on the level of the five-dimensional action. The vector sector of the reduced theory is encoded by a non-standard potential due to the Green-Schwarz term in six dimensions. This Green-Schwarz term also contains higher curvature couplings which are considered to establish the full map between anomaly coefficients and geometry. F-/M-theory duality is exploited by moving to the five-dimensional Coulomb branch after circle reduction and integrating out massive vector multiplets and matter hypermultiplets. The associated fermions then generate additional Chern-Simons couplings at one-loop. Further couplings involving the graviphoton are induced by quantum corrections due to excited Kaluza-Klein modes. On the M-theory side integrating out massive fields corresponds to resolving the singularities of the Calabi-Yau threefold, and yields intriguing relations between six-dimensional anomalies and classical topology.Comment: 55 pages, v2: typos corrected, discussion of loop corrections improve