Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum

Citation: Tabunoki, H., Gorman, M. J., Dittmer, N. T., & Kanost, M. R. (2016). Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum. Scientific Reports, 6, 8. doi:10.1038/srep29583Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails

Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum

Citation: Tabunoki, H., Gorman, M. J., Dittmer, N. T., & Kanost, M. R. (2016). Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum. Scientific Reports, 6, 8. doi:10.1038/srep29583Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails

Molecular Network Analysis Suggests Aberrant CREB-Mediated Gene Regulation in the Alzheimer Disease Hippocampus

The pathogenesis of Alzheimer disease (AD) involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs) and 559 incipient AD-related genes (IADGs) identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004). By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB) as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify thein silicoobservationsin vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB) antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD) in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.</jats:p

The metabolic ability of swallowtails results in the production of bioactive substances from plant components

Host plant selection may depend on the metabolic system in herbivorous insects. Although oligophagous insects take up specific host plant components, how host plant components and their biological activities are altered through their metabolic systems remains unknown. Here, by examining gene expression of metabolic enzymes and components in the larval frass, we investigated the metabolic ability of Papilio memnon larvae fed with Citrus x paradisi (grapefruit) against host plant components. The gene expression levels of some metabolic enzymes were fluctuated between the larval midgut and the larval fat bodies. Furthermore, the chloroform extract from the larval frass, but not that from grapefruit leaves, inhibited cell viability of human pancreatic cancer cell line, MIA PaCa2. Finally, we identified two chlorophyll catabolites, pheophorbide-a and pyropheophorbide-a, in the larval frass extract. Pyropheophorbide-a reduces cell viability of and induces morphological changes in cells of MIA PaCa2; in addition, pheophorbide-a and pyropheophorbide-a inhibit the aggregation of amyloid β-protein (human, 1–42). Therefore, the chemical structure and biological activity of host plant components are affected by the P. memnon metabolic system. Our findings may contribute to the understanding of the process for producing pheophorbide-a and pyrophephorbide-a from chlorophyll, facilitated by the metabolic ability of P. memnon larvae.journal articl

Molecular Network Analysis of T-Cell Transcriptome Suggests Aberrant Regulation of Gene Expression by NF-<i>κ</i>B As a Biomarker for Relapse of Multiple Sclerosis

Molecular mechanisms responsible for acute relapse of multiple sclerosis (MS) remain currently unknown. The aim of this study is to identify the relapse-specific biomarker genes in T lymphocytes of relapsing-remitting MS (RRMS). Total RNA of CD3+T cells isolated from six RRMS patients taken at the peak of acute relapse and at the point of complete remission was processed for DNA microarray analysis. We identified a set of 43 differentially expressed genes (DEG) between acute relapse and complete remission. By using 43 DEG as a discriminator, hierarchical clustering separated the cluster of relapse from that of remission. The molecular network of 43 DEG investigated by KeyMolnet, a bioinformatics tool for analyzing molecular interaction on the curated knowledge database, showed the most significant relationship with aberrant regulation of gene expression by the nuclear factor-kappa B (NF-κB) in T cells during MS relapse. These results support the logical hypothesis that NF-κB plays a central role in triggering molecular events in T cells responsible for induction of acute relapse of MS, and suggest that aberrant gene regulation by NF-κB on T-cell transcriptome might serve as a molecular biomarker for monitoring the clinical disease activity of MS.</jats:p

Dystrophic neurites express C9orf72 in Alzheimer's disease brains

INTRODUCTION: Chromosome 9 open reading frame 72 (C9orf72) is an evolutionarily conserved protein with unknown function, expressed at high levels in the brain. An expanded hexanucleotide GGGGCC repeat located in the first intron of the C9orf72 gene represents the most common genetic cause of familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Previous studies by immunohistochemistry with two different anti-C9orf72 antibodies named sc-138763 and HPA023873 showed that C9orf72 is expressed chiefly in the cytoplasm of neurons, and is concentrated in the synaptic terminals in the brains of FTD/ALS with or without C9orf72 repeat expansion as well as those of controls. At present, a pathological role of C9orf72 in the process of neurodegeneration remains unknown. METHODS: Using immunohistochemistry we studied C9orf72 expression in the frontal cortex and the hippocampus of six Alzheimer's disease (AD) and 13 control cases, including ALS, Parkinson's disease, multiple system atrophy, and non-neurological cases. RESULTS: The HPA023873 antibody showed a cross-reactivity to glial fibrillary acidic protein, and therefore stained intensely reactive astrocytes in AD and non-AD brains. Both sc-138763 and HPA023873 antibodies labeled the neuronal cytoplasm and the neuropil with variable intensities, and intensely stained a cluster of p62-negative, UBQLN1-positive swollen neurites, which were distributed in the CA1 region and the molecular layer in the hippocampus of both AD and non-AD brains. Most notably, both of these antibodies reacted strongly with dystrophic neurites accumulated on senile plaques in AD brains. CONCLUSION: These results suggest a general role of C9orf72 in the process of neurodegeneration in a range of human neurodegenerative diseases

Integrated effects of thermal acclimation and challenge temperature on cellular immunity in the plusiine moth larvae Chrysodeixis eriosoma (Lepidoptera: Noctuidae)

Temperature is one of the most influential factors for animals. The acclimation (rearing) and challenge temperatures are often more important than the given temperature per se. These effects on physiological responses have been known, but not well understood on immune responses. Here, we investigated the integrated effects of rearing and challenge temperatures on haemocyte populations in larvae of a plusiine moth, Chrysodeixis eriosoma. We hypothesize that the haemocyte concentration is decreased (increased) at higher (lower) temperatures from rearing temperatures and that the proportions of haemocyte types exhibit directional changes at higher (lower) temperatures to compensate for immune reactions. We expect that increasing (decreasing) the challenge temperature from the rearing temperature enhances (reduces) phagocytic activity. We found that higher temperatures slightly decreased the haemocyte concentration. We detected small changes in the proportions of haemocyte types among rearing temperatures, but the changes were non‐directional and most of them were statistically insignificant. We also found the integrated effects only with increases in the challenge temperatures, which resulted in increased phagocytosis, whereas no apparent reactions were detected with decreases in the challenge temperatures. Our results show that the haemocyte concentration is significantly affected by the rearing temperature, which implies that haematopoiesis depends on the ambient temperature.We discuss some adaptive and non‐adaptive components for the positive integrated effects of increases in the challenge temperatures. We also discussed the obtained non‐responsiveness in the integrated effects with decreases in the challenge temperatures

Comprehensive analysis of human microRNA target networks

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) mediate posttranscriptional regulation of protein-coding genes by binding to the 3' untranslated region of target mRNAs, leading to translational inhibition, mRNA destabilization or degradation, depending on the degree of sequence complementarity. In general, a single miRNA concurrently downregulates hundreds of target mRNAs. Thus, miRNAs play a key role in fine-tuning of diverse cellular functions, such as development, differentiation, proliferation, apoptosis and metabolism. However, it remains to be fully elucidated whether a set of miRNA target genes regulated by an individual miRNA in the whole human microRNAome generally constitute the biological network of functionally-associated molecules or simply reflect a random set of functionally-independent genes.</p> <p>Methods</p> <p>The complete set of human miRNAs was downloaded from miRBase Release 16. We explored target genes of individual miRNA by using the Diana-microT 3.0 target prediction program, and selected the genes with the miTG score ≧ 20 as the set of highly reliable targets. Then, Entrez Gene IDs of miRNA target genes were uploaded onto KeyMolnet, a tool for analyzing molecular interactions on the comprehensive knowledgebase by the neighboring network-search algorithm. The generated network, compared side by side with human canonical networks of the KeyMolnet library, composed of 430 pathways, 885 diseases, and 208 pathological events, enabled us to identify the canonical network with the most significant relevance to the extracted network.</p> <p>Results</p> <p>Among 1,223 human miRNAs examined, Diana-microT 3.0 predicted reliable targets from 273 miRNAs. Among them, KeyMolnet successfully extracted molecular networks from 232 miRNAs. The most relevant pathway is transcriptional regulation by transcription factors RB/E2F, the disease is adult T cell lymphoma/leukemia, and the pathological event is cancer.</p> <p>Conclusion</p> <p>The predicted targets derived from approximately 20% of all human miRNAs constructed biologically meaningful molecular networks, supporting the view that a set of miRNA targets regulated by a single miRNA generally constitute the biological network of functionally-associated molecules in human cells.</p

Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNβ-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNβ-related adverse effects in multiple sclerosis

BACKGROUND: A substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNβ) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNβ-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNβ-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects. METHODS: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis. RESULTS: Among 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils. CONCLUSION: IFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo