Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.     

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.     

Rutin modulates ASC expression in NLRP3 inflammasome: a study in alcohol and cerulein-induced rat model of pancreatitis

Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 µg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC–NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH–Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC–NLRP3 which might result in the reduced activation of caspase-1 and controlled cytokine production.     

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage,Sf101, from a serotype 7a strain

Background

Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of Rha^III, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.

Results

In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.

Conclusions

This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-742) contains supplementary material, which is available to authorized users.     

Effects of sequential depositional basins on lake response to urban and agricultural pollution: a palaeoecological analysis of the Qu'Appelle Valley, Saskatchewan, Canada

1. Palaeolimnological analyses of fossil diatoms and pigments were conducted in four lakes of the Qu'Appelle Valley, Saskatchewan, Canada, to quantify the effect of upstream depositional basins on lake response to urban and agricultural human activities. Pasqua, Echo, Mission and Katepwa lakes exhibit similar modern limnological characteristics, lie sequentially downstream from urban point sources of growth‐limiting nitrogen (N), yet drain similarly large areas of farmland (38–40 × 10³ km²). 2. Analyses indicated that all lakes were naturally productive, contained eutrophic diatoms (i.e. Stephanodiscus niagarae, S. hantzchii, S. parvus and Aulacoseira granulata), and supported blooms of colonial (as myxoxanthophyll) and potentially toxic N‐fixing cyanobacteria (aphanizophyll), even prior to the onset of European settlement (ca. 1890) and urban development (ca. 1930). 3. The onset of agricultural practices ca. 1890 had only modest effects on algal communities in the Qu'Appelle lakes, with subtle increases in eutrophic diatom species (Pasqua, Mission and Katepwa lakes) and 25–50% increases in pigment‐inferred algal abundance (Echo, Mission and Katepwa lakes). 4. Despite naturally high production, total algal abundance (β‐carotene) in upstream Pasqua Lake increased by more than 350% after intense urbanization beginning ca. 1930, while eutrophic diatoms became more common and cyanobacteria populations increased ten‐fold. Principal components analysis (PCA) explained 64% of diatom variance, and identified three eras corresponding to baseline, pre‐agricultural communities (1776–1890), an era of high production (ca. 1925–1960) and recent variable community composition following tertiary treatment of urban sewage (ca. 1977–1990). 5. Analyses of three downstream lakes demonstrated that urban impacts following 1930 remained evident in fossil profiles of β‐carotene and myxoxanthophyll, but that large blooms of N‐fixing cyanobacteria were restricted to the past 25 years at downstream Mission and Katepwa lakes. Similarly, PCA showed that fossil diatom assemblages exhibited little directional variation until the 1970s. 6. Together, these analyses support the hypothesis that upstream lakes were effective at reducing the impacts of point‐source urban nutrients on downstream lakes. In contrast, diffuse agricultural activities had only limited impacts on water quality and these were less well ameliorated by upstream basins.     

New perspectives of nanoneuroprotection, nanoneuropharmacology and nanoneurotoxicity: modulatory role of amino acid neurotransmitters, stress, trauma, and co-morbidity factors in nanomedicine

Recent advancement in nanomedicine suggests that nanodrug delivery using nanoformulation of drugs or use of nanoparticles for neurodiagnostic and/or neurotherapeutic purposes results in superior effects than the conventional drugs or parent compounds. This indicates a bright future for nanomedicine in treating neurological diseases in clinics. However, the effects of nanoparticles per se in inducing neurotoxicology by altering amino acid neurotransmitters, if any, are still being largely ignored. The main aim of nanomedicine is to enhance the drug availability within the central nervous system (CNS) for greater therapeutic successes. However, once the drug together with nanoparticles enters into the CNS compartments, the fate of nanomaterial within the brain microenvironment is largely remained unknown. Thus, to achieve greater success in nanomedicine, our knowledge in understanding nanoneurotoxicology in detail is utmost important. In addition, how co-morbidity factors associated with neurological disease, e.g., stress, trauma, hypertension or diabetes, may influence the neurotherapeutic potentials of nanomedicine are also necessary to explore the details. Recent research in our laboratory demonstrated that engineered nanoparticles from metals or titanium nanowires used for nanodrug delivery in laboratory animals markedly influenced the CNS functions and alter amino acid neurotransmitters in healthy animals. These adverse reactions of nanoparticles within the CNS are further aggravated in animals with different co-morbidity factors viz., stress, diabetes, trauma or hypertension. This effect, however, depends on the composition and dose of the nanomaterials used. On the other hand, nanodrug delivery by TiO₂ nanowires enhanced the neurotherapeutic potential of the parent compounds in CNS injuries in healthy animals and do not alter amino acids balance. However, in animals with any of the above co-morbidity factors, high dose of nanodrug delivery is needed to achieve some neuroprotection. Taken together, it appears that while exploring new nanodrug formulations for neurotherapeutic purposes, co-morbidly factors and composition of nanoparticles require more attention. Furthermore, neurotoxicity caused by nanoparticles per se following nanodrug delivery may be examined in greater detail with special regards to changes in amino acid balance in the CNS.     

miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2

In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.     

Paclobutrazol Arrests Vegetative Growth and Unveils Unexpressed Yield Potential of Jatropha curcas

Although the process for making EN 14214 grade Jatropha methyl ester (biodiesel) capable of running unmodified diesel engines in neat form has been demonstrated, getting higher seed yield from Jatropha shrubs in wastelands is critical to the success of Jatropha biodiesel. But, low productivity is inherent to many Jatropha curcas germplasms and raising large-scale plantations using such untested planting material can lead to wasteful expenditures. Unreliable and poor flowering and fruiting are important factors responsible for low productivity in the species. Although much is known about growth retardants applied to field and horticultural crops, their role in improving the seed productivity of Jatropha has never been explored. Here we report for the first time that paclobutrazol could be an extremely useful chemical, whose dose and time of application, if optimized, can significantly reduce unwanted vegetative growth, with concomitant improvement in yield and seed oil content of Jatropha. In the year following application of paclobutrazol, an unexpected increase in seed yield, as high as 1127% relative to controls, was obtained from one such unproductive Jatropha germplasm. We hypothesize that low seed production in this species may be a result of excess vegetative growth caused by an unfavorable endogenous hormonal configuration which competes with growth and development of flower, fruit, or seed. This undesired physiological state can be reversed by paclobutrazol application to achieve maximum oil yield from this energy shrub that holds great promise in the future.     

Randomized single oral dose phase 1 study of safety,tolerability, and pharmacokinetics of Iminosugar UV-4 Hydrochloride (UV-4B) in healthy subjects

BackgroundUV-4 (N-(9’-methoxynonyl)-1-deoxynojirimycin, also called MON-DNJ) is an iminosugar small-molecule oral drug candidate with in vitro antiviral activity against diverse viruses including dengue, influenza, and filoviruses and demonstrated in vivo efficacy against both dengue and influenza viruses. The antiviral mechanism of action of UV-4 is through inhibition of the host endoplasmic reticulum-resident α-glucosidase 1 and α-glucosidase 2 enzymes. This inhibition prevents proper glycan processing and folding of virus glycoproteins, thereby impacting virus assembly, secretion, and the fitness of nascent virions.Methodology/Principal findingsHere we report a first-in-human, single ascending dose Phase 1a study to evaluate the safety, tolerability, and pharmacokinetics of UV-4 hydrochloride (UV-4B) in healthy subjects (ClinicalTrials.gov Identifier {"type":"clinical-trial","attrs":{"text":"NCT02061358","term_id":"NCT02061358"}}NCT02061358). Sixty-four subjects received single oral doses of UV-4 as the hydrochloride salt equivalent to 3, 10, 30, 90, 180, 360, 720, or 1000 mg of UV-4 (6 subjects per cohort), or placebo (2 subjects per cohort). Single doses of UV-4 hydrochloride were well tolerated with no serious adverse events or dose-dependent increases in adverse events observed. Clinical laboratory results, vital signs, and physical examination data did not reveal any safety signals. Dose-limiting toxicity was not observed; the maximum tolerated dose of UV-4 hydrochloride in humans has not yet been determined (>1000 mg). UV-4 was rapidly absorbed and distributed after dosing with the oral solution formulation used in this study. Median time to reach maximum plasma concentration ranged from 0.5–1 hour and appeared to be independent of dose. Exposure increased approximately in proportion with dose over the 333-fold dose range. UV-4 was quantifiable in pooled urine over the entire collection interval for all doses.Conclusions/SignificanceUV-4 is a host-targeted broad-spectrum antiviral drug candidate. At doses in humans up to 1000 mg there were no serious adverse events reported and no subjects were withdrawn from the study due to treatment-emergent adverse events. These data suggest that therapeutically relevant drug levels of UV-4 can be safely administered to humans and support further clinical development of UV-4 hydrochloride or other candidate antivirals in the iminosugar class.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02061358","term_id":"NCT02061358"}}NCT02061358 https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT02061358","term_id":"NCT02061358"}}NCT02061358.