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Cockroaches live in places where various pathogens exist. Thus, they are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the Periplaneta americana transcriptome and detected periplanetasin-5 using an in silico antimicrobial peptide prediction method. In this study, we investigated whether periplanetasin-5 has anticancer activity against the human leukemia cell line K562. Cell growth and survival of K562 cells treated with periplanetasin-5 were decreased in a dose-dependent manner. By using flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) staining, and DNA fragmentation, we found that periplanetasin-5 induced apoptotic and necrotic cell death in leukemia cells. In addition, these events were associated with increased levels of the proapoptotic proteins Fas and cytochrome c and reduced levels of the anti-apoptotic protein Bcl-2. Periplanetasin-5 induces the cleavage of pro-caspase-9, pro-caspase-8, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP). The above data suggest that periplanetasin-5 induces apoptosis via both the intrinsic and extrinsic pathways. Furthermore, caspase-related apoptosis was further confirmed by using the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), which reversed the periplanetasin-5-induced reduction in cell viability. In conclusion, periplanetasin-5 caused apoptosis in leukemia cells, suggesting its potential utility as an anticancer therapeutic agent.

Evolutionary immune system upgradation is done by all living organism to survive from the invading pathogens. Insects has a strong defense system comprising of anatomical barrier, humoral antimicrobial peptide (AMP) production and cellular immune components. Locusta migratoria (L. migratoria) is an agricultural pest insect migrating long distance might encounter distinct pathogens. Owing to this, we aimed in identifying AMPs present in the L. migratoria and elucidate the antimicrobial activity. The migratory locust was immunized with Escherichia coli, Staphylococcus aureus, Candida albicans mixture, then RNA was isolated and sequenced. The L. migratoria transcriptome was de novo assembled using trinity and screened for AMPs propensity specifically molecular and aggregation properties. The novelty is tested by blasting in known AMP databases. Finally, obtained novel putative AMPs were then tested for antimicrobial and hemolytic activity. The prediction resulted in 3,524 putative AMPs which was further screened down to ten AMPs for final testing. Two peptides showing promising antimicrobial effects were obtained. All the analyzed peptides showed no hemolysis confirms its non-toxicity. Thus, our peptides could be promising drug candidate and can be used as an alternative to antibacterial or antifungal therapy with further validations.

Previously, we performed an in silico analysis of the Periplaneta americana transcriptome. Antimicrobial peptide candidates were selected using an in silico antimicrobial peptide prediction method. It was found that periplanetasin-5 had antimicrobial activity against yeast and grampositive and gram-negative bacteria. In the present study, we demonstrated the anti-inflammatory activities of periplanetasin-5 in mouse macrophage Raw264.7 cells. No cytotoxicity was observed at 60 μg/ml periplanetasin-5, and treatment decreased nitric oxide production in Raw264.7 cells exposed to lipopolysaccharide (LPS). In addition, quantitative RT-PCR and enzyme-linked immunosorbent assay revealed that periplanetasin-5 reduced cytokine (tumor necrosis factor-α, interleukin-6) expression levels in the Raw264.7 cells. Periplanetasin-5 controlled inflammation by inhibiting phosphorylation of MAPKs, an inflammatory signaling element, and reducing the degradation of IκB. Through LAL assay, LPS toxicity was found to decrease in a periplanetasin-5 dose-dependent manner. Collectively, these data showed that periplanetasin-5 had antiinflammatory activities, exemplified in LPS-exposed Raw264.7 cells. Thus, we have provided a potentially useful antibacterial peptide candidate with anti-inflammatory activities.

In a previous work, we performed de novo RNA sequencing of Allomyrina dichotoma using next generation sequencing and identified several antimicrobial peptide candidates based on transcriptome analysis. Among them, a cationic antimicrobial peptide, allomyrinasin, was selected bioinformatically based on its physicochemical properties. Here, we assessed the antimicrobial and anti-inflammatory activities of allomyrinasin against microorganisms and mouse macrophage Raw264.7 cells. Allomyrinasin showed antimicrobial activities against various microbes and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Furthermore, quantitative RT-PCR and ELISA revealed that allomyrinasin reduced cytokine expression levels in the Raw264.7 cells. We also identified inducible nitric oxide synthase, cyclooxygenase-2 expression, and PGE2 production through western blot analysis and ELISA. We confirmed that allomyrinasin bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. Taken together, these data indicate that allomyrinasin has antimicrobial and anti-inflammatory activities as exemplified in lipopolysaccharide-induced Raw264.7 cells. We have provided a potentially useful antimicrobial peptide candidate that has both antimicrobial and anti-inflammatory activities.

Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the

Background & Aims Patients with or without cancers who undergo major gastrointestinal surgery suffer from malnutrition due to their catabolic status during the postoperative period. In this study, we evaluated the effect of the clinical application of protein-enhanced diet using mealworms in patients who underwent hepato-pancreato-biliary surgeries. Methods This study was designed as a prospective, two-armed, and double-blinded phase III study. The target number of enrolled patients was 216, and the patients were randomized on a 1:1 basis, either to the trial group (consuming mealworms) or to the control group (consuming grain powder). The primary endpoint was to examine the changes in body composition, including phase angle. For secondary outcomes, the activities of immune cells were evaluated using the patients’ blood samples. Results No difference in the demographic characteristics of patients was observed. The ratio of the actual protein intake to the recommended daily intake in the trial group was significantly higher than that in the control group (110.03% vs. 98.80%, p=0.023). In the data on body composition measured by InBody S-10, the ratios in body cell mass, fat free mass, muscle mass, and phase angle at the study endpoint compared to those at admission showed no statistically significant difference between the two groups. Immune cell analyses suggested that cytotoxic T cells in the trial group had higher activity than in the study group (1.192 vs. 0.974, p=0.028). Conclusions In this study, protein-enhanced diet using mealworms clinically improved the activity of immune cells. However, it did not significantly improve the patients’ nutritional status after they experienced hepato-pancreato-biliary surgeries.

Antimicrobial peptides (AMPs) are the frontline innate defense system evolutionarily preserved in insects to combat invading pathogens. These AMPs could serve as an alternative to classical antibiotics to overcome the burden of treating multidrug resistant bacteria. Psacotheasin, a knottin type AMP was isolated from Psacothea hilaris and shown to exhibit antimicrobial activity, especially against fungi through apoptosis mediated cell death. In this study, we aimed to identify novel probable AMPs from Psacothea hilaris, the yellow spotted longicorn beetle. The beetle was immunized with the two bacterial strains (E. coli and S. aureus), and the yeast strain C. albicans. After immunization, total RNA was isolated and sequenced in Illumina platform. Then, beetle transcriptome was de novo assembled and searched for putative AMPs with the known physiochemical features of the AMPs. A selection of AMP candidates were synthesized and tested for antimicrobial activity. Four peptides showed stronger activity against E. coli than the control AMP, melittin while one peptide showed similar activity against S. aureus. Moreover, four peptides and two peptides showed antifungal activity stronger than and similar to melittin, respectively. Collectively one peptide showed both antibacterial and antifungal activity superior to melittin, thus, it provides a potent antimicrobial peptide. All the peptides showed no hemolysis in all the tested concentrations. These results suggest that in silico mining of insects&rsquo, transcriptome could be a promising tool to obtain and optimize novel AMPs for human needs.

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.

Given the challenges posed by antibiotic resistant microbes and the high mortality rate associated with sepsis, there is an urgent need to develop novel peptide antibiotics that exhibit both antimicrobial and anti-inflammatory activities. Herein, we evaluated antimicrobial activity and anti-inflammatory activity of psacotheasin 2, one of the antimicrobial peptide candidates identified previously using an in silico analysis on the transcriptome of Psacothea hilaris. In addition to exhibiting antimicrobial activities against microorganisms without inducing hemolysis, psacotheasin 2 also decreased the nitric oxide production in lipopolysaccharide (LPS)-induced Raw264.7 cells. Moreover, ELISA and western blot analysis revealed that psacotheasin 2 reduced the expression levels of pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Further, we found that psacotheasin 2 markedly reduced the expression levels of pro-inflammatory cytokines (IL-6 and IL-1β) by regulating mitogen-activated protein kinases (MAPKs) and nuclear factor-kB (NF-kB) signaling in LPS-induced Raw264.7 cells. We also confirmed that the binding of psacotheasin 2 to bacterial cell membranes occurs via a specific interaction with LPS. In mouse models of LPS-induced shock, psacotheasin 2 significantly enhanced the survival rate and recovered weight by attenuating pro-inflammatory cytokines. Thus, psacotheasin 2 could be a promising candidate as a peptide antiseptic agent.

Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN-mediated autophagy is a complex process involving many autophagy-regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large-scale yeast-human two-hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN-Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule-associated protein light chain 3-EGFP to autophagosomes. In N2a cells, OPTN-induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN-Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation-induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a-induced autophagy. These results show that the OPTN-Rab1a complex modulates autophagosome formation in neuroblastoma cells.

In a previous work, we performed

Insect antimicrobial peptides (AMPs) have a wide range of functions and potential applications, and have recently attracted attention as alternative foods and medicines for humans. Our study performed transcriptome analysis to explore the potential of the red-striped golden stink bug (Poecilocoris lewisi), and as a result, we have discovered new features of P. lewisi that have not been identified. Specifically, defensin found in P. lewisi is a well-known AMP and is expressed by various plants, animals and fungi for host defense. Moreover, the discovery of defensin in P. lewisi provides new research and important information. In this study, we identified AMP and related DEG in P. lewisi that are closely related to human disease and immune response. These findings will provide the basis and important information for future research on P. lewisi that has not yet been studied.

Recently, unbiased functional genetic selection identified novel cell migration-regulating genes. This RNAi-based functional selection was performed using 63,996 pooled lentiviral shRNAs targeting 21,332 mouse genes. After five rounds of selection using cells with accelerated or impaired migration, shRNAs were retrieved and identified by half-hairpin barcode sequencing using cells with the selected phenotypes. This selection process led to the identification of 29 novel cell migration regulators. One of these candidates, anaplastic lymphoma kinase (ALK), was further investigated. Subsequent studies revealed that ALK promoted cell migration through the PI3K-AKT pathway via the p55γ regulatory subunit of PI3K, rather than more commonly used p85 subunit. Western blot and immunohistochemistry studies using mouse brain tissues revealed similar temporal expression patterns of ALK, phospho-p55γ, and phospho-AKT during different stages of development. These data support an important role for the p55γ subunit of PI3K in ALK-induced cell migration during brain development.

Hypothalamic endoplasmic reticulum (ER) stress is known to be increased in obesity. Induction of ER stress on hypothalamic neurons has been reported to cause hypothalamic neuronal apoptosis and malfunction of energy balance, leading to obesity. Carbenoxolone is an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor that converts inactive glucocorticoid into an active form. In addition to its metabolic effect via enzyme inhibitory action, carbenoxolone has shown anti-apoptotic activity in several studies. In this study, the direct effects of carbenoxolone on ER stress and cell death in hypothalamic neurons were investigated. Carbenoxolone attenuated tunicamycin induced ER stress-mediated molecules such as spliced XBP1, ATF4, ATF6, CHOP, and ROS generation. In vivo study also revealed that carbenoxolone decreased tunicamycin-induced ER stress in the hypothalamus. In conclusion, the results of this study show that carbenoxolone has protective effects against tunicamycin induced-ER stress and apoptosis in hypothalamic neurons, suggesting its direct protective effects against obesity. Further study is warranted to clarify the effects of carbenoxolone on hypothalamic regulation of energy balance in obesity.

We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

mTORC1 plays a key role in autophagy as a negative regulator. The currently known targets of mTORC1 in the autophagy pathway mainly function at early stages of autophagosome formation. Here, we identify that mTORC1 inhibits later stages of autophagy by phosphorylating UVRAG. Under nutrient-enriched conditions, mTORC1 binds and phosphorylates UVRAG. The phosphorylation positively regulates the association of UVRAG with RUBICON, thereby enhancing the antagonizing effect of RUBICON on UVRAG-mediated autophagosome maturation. Upon dephosphorylation, UVRAG is released from RUBICON to interact with the HOPS complex, a component for the late endosome and lysosome fusion machinery, and enhances autophagosome and endosome maturation. Consequently, the dephosphorylation of UVRAG facilitates the lysosomal degradation of epidermal growth factor receptor (EGFR), reduces EGFR signaling, and suppresses cancer cell proliferation and tumor growth. These results demonstrate that mTORC1 engages in late stages of autophagy and endosome maturation, defining a broader range of mTORC1 functions in the membrane-associated processes.

Periodontitis and insulin resistance (IR) show bidirectional relationship. No studies have assessed the associations of periodontitis with IR, impaired β-cell function, and impaired fasting glucose (IFG) in the general population. We investigated these associations in a representative sample of the Korean population. The subjects were 8,248 males and 10,874 females, who were ≥ 20 years of age and participants in the third, fourth, and fifth Korea National Health and Nutritional Examination Surveys (2008-2010). Periodontitis was defined as community periodontal index (CPI) ≥ code 3 according to World Health Organization criteria. Homeostasis model assessments of IR and β-cell function (HOMA-IR and HOMA-β) were calculated. Participants with periodontitis showed a higher prevalence of diabetes than those without periodontitis. Among subjects without diabetes, after adjustment for confounding factors including age, gender, body mass index, systolic blood pressure, serum total cholesterol, smoking status, alcohol consumption, region, and regular exercise, a comparison of participants with periodontitis vs those without showed a significantly higher prevalence of IFG (28.5% vs. 17.7%, p

Recently, we reported that Lumbricusin, an antimicrobial peptide isolated from earthworm Lumbricus terrestris, enhanced neuronal proliferation and ameliorated motor dysfunction and dopaminergic neurodegeneration. Accumulating evidence suggests that neurodegeneration is the primary pathological feature of acute or chronic inflammation mediated by microglia, the resident macrophage of the central nervous system. Therefore, microglial activation inhibitors may be useful as therapeutic agents for neurodegenerative diseases. To determine whether Lumbricusin ameliorates neuroinflammation through inhibition of microglial activation by lipopolysaccharides (LPS), we newly synthesized 9-mer Lumbricusin analogues based on the amino acid sequence of Lumbricusin. One of these, Lumbricusin Analogue 5 (LumA5; QLICWRRFR-NH2), markedly reduced expression of enzymes (COX-2, iNOS), cytokines (IL-6, IL-1β, TNF-α), and signal transduction factors (AKT, MAPKs, NF-κB) involved in inflammation triggered by LPS in vitro and in vivo. In addition, LumA5 inhibited the cytotoxicity of conditioned medium prepared by LPS-activated BV-2 microglia to neuronal SH-SY5Y cells and improved cell viability. These results indicate that LumA5 may be a potential therapeutic agent for the treatment of various neuroinflammatory conditions.

Despite the increasing interest in insect-based bioactive products, the biological activities of these products are rarely studied adequately. Larvae of Tenebrio molitor, the yellow mealworm, have been eaten as a traditional food and provide many health benefits. Therefore, we hypothesized that T. molitor larvae might influence adipogenesis and obesity-related disorders. In the present study, we investigated the anti-adipogenic and antiobesity effects of T. molitor larvae in vitro and in vivo. The lipid accumulation and triglyceride content in mature adipocytes was reduced significantly (up to 90%) upon exposure to an ethanol extract of T. molitor larvae, without a reduction in cell viability. Exposure also resulted in key adipogenic and lipogenic transcription factors. Additionally, in adipogenic differentiation medium the extract induced phosphorylation of adenosine monophosphate (AMP)-activated protein kinase and mitogen-activated protein kinases. Daily oral administration of T. molitor larvae powder to obese mice fed high-fat diet attenuated body weight gain. We also found that the powder efficiently reduced hepatic steatosis as well as aspartate and alanine transaminase enzyme levels in mice fed a high-fat diet. Our results suggest that T. molitor larvae extract has an antiobesity effect when administered as a food supplement and has potential as a therapeutic agent for obesity.

Lipocalin-2 (LCN2) plays an important role in cellular processes as diverse as cell growth, migration/invasion, differentiation, and death/survival. Furthermore, recent studies indicate that LCN2 expression and secretion by glial cells are induced by inflammatory stimuli in the central nervous system. The present study was undertaken to examine the regulation of LCN2 expression in experimental autoimmune encephalomyelitis (EAE) and to determine the role of LCN2 in the disease process. LCN2 expression was found to be strongly increased in spinal cord and secondary lymphoid tissues after EAE induction. In spinal cords astrocytes and microglia were the major cell types expressing LCN2 and its receptor 24p3R, respectively, whereas in spleens, LCN2 and 24p3R were highly expressed in neutrophils and dendritic cells, respectively. Furthermore, disease severity, inflammatory infiltration, demyelination, glial activation, the expression of inflammatory mediators, and the proliferation of MOG-specific T cells were significantly attenuated in Lcn2-deficient mice as compared with wild-type animals. Myelin oligodendrocyte glycoprotein-specific T cells in culture exhibited an increased expression of Il17a, Ifng, Rorc, and Tbet after treatment with recombinant LCN2 protein. Moreover, LCN2-treated glial cells expressed higher levels of proinflammatory cytokines, chemokines, and MMP-9. Adoptive transfer and recombinant LCN2 protein injection experiments suggested that LCN2 expression in spinal cord and peripheral immune organs contributes to EAE development. Taken together, these results imply LCN2 is a critical mediator of autoimmune inflammation and disease development in EAE and suggest that LCN2 be regarded a potential therapeutic target in multiple sclerosis.

We recently reported the in vitro and in vivo antiobesity effects of Tenebrio molitor larvae, a traditional food in many countries, but it remains unknown how the larvae affect appetite regulation in mice with diet-induced obesity. We hypothesized that the extract of T molitor larvae mediates appetite by regulating neuropeptide expression. We investigated T molitor larvae extract's (TME's) effects on anorexigenesis and endoplasmic reticulum (ER) stress-induced orexigenic neuropeptide expression in the hypothalami of obese mice. Intracerebroventricular TME administration suppressed feeding by down-regulating the expression of the orexigenic neuropeptides neuropeptide Y and agouti-related protein. T molitor larvae extract significantly reduced the expression of ER stress response genes. These results suggest that TME and its bioactive components are potential therapeutics for obesity and ER stress-driven disease states.

Glial cells are non-neuronal components of the central nervous system (CNS). They are endowed with diverse functions and are provided with tools to detect their own activities and those of neighboring neurons. Glia and neurons are in continuous reciprocal communication under both physiological and neuropathological conditions, and glia secrete various guidance factors or proteinaceous signals that service vital neuronal-glial interactions in health and disease. Analysis and profiling of glial secretome, especially of microglia and astrocytes, have raised new expectations for the diagnosis and treatment of CNS disorders, and the availability of a catalog of glia-secreted proteins might provide an origin for further research on the complex extracellular signaling mediated by glial cells. Components of the glial secretome play important roles as mediators and modulators of brain structure and function during neuroprotection and neurodegeneration. Therapeutic hypothermia has been acclaimed an effective modulator of brain injury via its substantial effect on the protein expression profiles of glia. Furthermore, emerging proteomic tools and methodologies make feasible the documentation of the reactive glial secretome signature. This review focuses on reactive glial cells and the uniqueness of their secretome during diverse neuropathological conditions. This article is part of a Special Issue entitled: An Updated Secretome.

Therapeutic hypothermia is a robust therapeutic tool in experimental stroke models but its clinical applications are limited. Furthermore, optimal conditions for therapeutic hypothermia, such as, temperature and the initiation and duration of cooling must be individualized. Here, we evaluated the therapeutic effects of delayed local hypothermia, administered for 44 hr after 4 hr of reperfusion in a rat model of transient middle cerebral artery occlusion (tMCAo), using a cooling device that allowed controlled local hypothermia (31 °C) in brain. Histological data revealed that local hypothermia significantly reduced infarct volumes and glial hypertrophic activation. Brain water contents, IgG leakage, and Evans Blue extravasation were notably reduced by local hypothermia. Furthermore, local hypothermia had strong vasculoprotective effects, as determined by immunohistochemistry and Western blot analyses for endothelial barrier antigen (EBA), laminin, aquaporin-4, and tight junction proteins in brain. Our data indicate that delayed/prolonged local hypothermia confers neurovascular protection, reduces brain edema, and inhibits inflammatory glial activation, and suggest that hypothermic conservation of vascular structures and functions account for the therapeutic effects of local hypothermia observed in this model of experimental stroke.

Therapeutic hypothermia is a useful intervention against brain injury in experimental models and patients, but its therapeutic applications are limited due to its ill-defined mode of action. Glia cells maintain homeostasis and protect the central nervous system from environmental change, but after brain injury, glia are activated and induce glial scar formation and secondary injury. On the other hand, therapeutic hypothermia has been shown to modulate glial hyperactivation under various brain injury conditions. We considered that knowledge of the effect of hypothermia on the molecular profiles of glia and on their phenotypes would improve our understanding of the neuroprotective mechanism of hypothermia. Here, we review the findings of recent studies that examined the effect of hypothermia on proteome changes in reactive glial cells in vitro and in vivo. The therapeutic effects of hypothermia are associated with the inhibition of reactive oxygen species generation, the maintenance of ion homeostasis, and the protection of neurovascular units in cultured glial cells. In an animal model, a distinct pattern of protein alterations was detected in glia following hypothermia under ischemic/reperfusion conditions. In particular, hypothermia was found to exert a neuroprotective effect against ischemic brain injury by regulating specific glial signaling pathways, such as, glutamate signaling, cell death, and stress response, and by influencing neural dysfunction, neurogenesis, neural plasticity, cell differentiation, and neurotrophic activity. Furthermore, the proteins that were differentially expressed belonged to various pathways and could mediate diverse phenotypic changes of glia in vitro or in vivo. Therefore, hypothermia-modulated glial proteins and subsequent phenotypic changes may form the basis of the therapeutic effects of hypothermia.

Astrocytes provide physical and metabolic support for neurons in the central nervous system. Reactive astrocytes, however, play a crucial role in neuroinflammation after brain injury. Therapeutic hypothermia has been shown to be neuroprotective in brain injury patients. In order to understand the effect of therapeutic hypothermia on astrocytes in stroke patients, we performed a proteomic profiling of astrocytes following transient focal cerebral ischemia in rats under normothermic (37°C) or mild hypothermic condition (33°C). Primary astrocytes were prepared from rat brain using a Percoll gradient method, and their proteome profiles were determined by using LC/ESI-MS/MS followed by high-throughput label-free quantification. Comparison of proteome profiles of astrocytes following transient focal ischemia revealed that hypothermia upregulated (≥1.5-fold) 86 proteins and downregulated (≤0.6-fold) 47 proteins compared to normothermic condition. The changes of protein expression were validated by Western blot or RT-PCR. Ingenuity Pathways Analysis and Database for Annotation, Visualization and Integrated Discovery analyses of the up- or downregulated proteins indicate that hypothermia influences glutamatergic signaling, cell death, and stress response of astrocytes in the ischemic brain. Therefore, therapeutic hypothermia may be neuroprotective in stroke patients by modulating astrocytic functions and survival.

Orosomucoid (ORM) is an acute-phase protein that belongs to the immunocalin subfamily, a group of small-molecule-binding proteins with immunomodulatory functions. Little is known about the role of ORM proteins in the CNS. The aim of the present study was to investigate the brain expression of ORM and its role in neuroinflammation. Expression of Orm2, but not Orm1 or Orm3, was highly induced in the mouse brain after systemic injection of lipopolysaccharide (LPS). Plasma levels of ORM2 were also significantly higher in patients with cognitive impairment than in normal subjects. RT-PCR, Western blot, and immunofluorescence analyses revealed that astrocytes are the major cellular sources of ORM2 in the inflamed mouse brain. Recombinant ORM2 protein treatment decreased microglial production of proinflammatory mediators and reduced microglia-mediated neurotoxicityin vitro. LPS-induced microglial activation, proinflammatory cytokines in hippocampus, and neuroinflammation-associated cognitive deficits also decreased as a result of intracerebroventricular injection of recombinant ORM2 proteinin vivo. Moreover, lentiviral shRNA-mediated Orm2 knockdown enhanced LPS-induced proinflammatory cytokine gene expression and microglial activation in the hippocampus. Mechanistically, ORM2 inhibited C-C chemokine ligand 4 (CCL4)-induced microglial migration and activation by blocking the interaction of CCL4 with C-C chemokine receptor type 5. Together, the results from our cultured glial cells, mouse neuroinflammation model, and patient studies suggest that ORM2 is a novel mediator of astrocyte–microglial interaction. We also report that ORM2 exerts anti-inflammatory effects by modulating microglial activation and migration during brain inflammation. ORM2 can be exploited therapeutically for the treatment of neuroinflammatory diseases.SIGNIFICANCE STATEMENTNeural cell interactions are important for brain physiology and pathology. Particularly, the interaction between non-neuronal cells plays a central role in regulating brain inflammation, which is closely linked to many brain disorders. Here, we newly identified orosomucoid-2 (ORM2) as an endogenous protein that mediates such non-neuronal glial cell interactions. Based on the critical role of astrocyte-derived ORM2 in modulating microglia-mediated neuroinflammation, ORM2 can be exploited for the diagnosis, prevention, or treatment of devastating brain disorders that have a strong neuroinflammatory component, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.

AIM Diabetes is a known risk factor for hearing impairment. No study regarding the association of insulin resistance (IR), β-cell dysfunction and impaired fasting glucose (IFG) with hearing impairment has been reported in the population without diabetes. We examined these associations using a representative sample of the Korean population. METHODS Participants included 1589 males and 2496 females, who were ≥ 20 years of age and without diabetes from the Korea National Health and Nutritional Examination Surveys of the Korean population (2010). Audiometric tests and laboratory examinations were performed. Homeostasis model assessments of IR and β-cell function (HOMA-IR and HOMA-β) were calculated. RESULTS The prevalence of both high- and low-/mid-frequency hearing impairment among subjects with IFG was higher compared with those with normal glycaemia (42.2% vs. 24.5%, 14.7% vs. 7.8%, respectively). After adjustment for age, HOMA-IR and HOMA-β showed significant association with high-frequency hearing impairment in males. In the multiple logistic regression analyses adjusting for confounding variables, the presence of IFG, higher HOMA-IR and lower HOMA-β remained as independent risk factors for high-frequency mild hearing impairment in males < 70 years [odds ratio (OR) 1.441, 95% confidence interval (95% CI), 1.056 to 1.967; OR, 1.448, 95% CI, 1.039 to 2.101; and OR, 0.447, 95% CI, 0.274 to 0.729, P < 0.05, respectively). CONCLUSIONS IR, β-cell dysfunction and IFG are associated with high-frequency mild hearing impairment in the male population < 70 years before the onset of diabetes.

Increased endoplasmic reticulum (ER) stress is known to be one of the causes of hypothalamic neuronal damage, as well as a cause of metabolic disorders such as obesity and diabetes. Recent evidence has suggested that Undaria pinnatifida (UP), an edible brown algae, has antioxidant activity. However, the neuroprotective effect of UP has yet to be examined. In this study, to investigate the neuroprotective effect of UP on ER stress-induced neuronal damage in mouse hypothalamic neurons, mice immortal hypothalamic neurons (GT1-7) were incubated with extract of UP. ER stress was induced by treating with tunicamycin. Tunicamycin induced apoptotic cell death was compared with the vehicle treatment through excessive ER stress. However UP protected GT1-7 cells from cell death, occurring after treatment with tunicamycin by reducing ER stress. Treatment with UP resulted in reduced increment of ATF6 and CHOP, and recovered the decrease of phosphorylation of Akt/mTOR by tunicamycin and the increment of autophagy. These results show that UP protects GT1-7 cells from ER stress induced cell death through the Akt/mTOR pathway. The current study suggests that UP may have a beneficial effect on cerebral neuronal degeneration in metabolic diseases with elevated ER stress.

ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling. ( 1-3) Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.

Phagocytosis is a critical host defense mechanism that clears invading pathogens, apoptotic cells, and cell debris; it is an essential process for normal development, tissue remodeling, immune response, and inflammation. Here, a functional selection strategy was used to isolate novel phagocytosis-promoting genes. After the retroviral transfer of mouse brain cDNA library into NIH3T3 mouse fibroblast cells, cell sorting was used to select the cells that phagocytosed fluorescent zymosan particles. The cDNAs were retrieved from the selected cells and identified by DNA sequencing as eIF5A, Meg3, Tubb5, Sparcl-1, Uchl-1, Bsg (CD147), Ube2v1, and Pamr1. The phagocytosis-promoting activity for some of these cDNAs was confirmed by transient transfection in the independent phagocytosis assays. Thus, the unbiased selection procedure successfully identified multiple phagocytosis-promoting genes. The selection method can be applied to other cell-based assays where cells with a desired phenotype can be physically separated. Moreover, the new gene targets uncovered in this study could be relevant to biomolecule screening in search of phagocytosis-regulating agents. In a small-scale screen, a series of imidazopyridine compounds was tested to identify the small molecules that modulate eIF5A-mediated phagocytic activity. Several compounds that influenced the phagocytic activity can be further used as chemical-genetic tools to delineate the mechanisms of eIF5A action and be potential drug candidates that are capable of therapeutically modulating phagocytic activity.

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K(+) channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC(20). Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.

Summary Acetylation of the mineralocorticoid receptor (MR) by inhibition of lysine deacetylases attenuates MR's transcriptional activity. However, the specific lysine acetyltransferases that are responsible for acetylation of the MR remain unknown. We hypothesized that the acetyltransferases cyclic adenosine monophosphate response element-binding binding protein (CBP) and acetyltransferase p300 (p300) attenuate transcriptional activity of the MR through its acetylation. Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes was analysed by chromatin immunoprecipitation. Acetylation of the MR was determined by western blot with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. In human embryonic kidney (HEK) 293 cells, overexpression of CBP or p300, but not p300/CBP-associated factor, increased MR acetylation and decreased expression of MR target genes. The downregulation of target genes coincided with a decrease in the recruitment of MR and Pol II to specific hormone response elements. These results demonstrate that overexpression of CBP or p300 attenuates the transcriptional activity of the MR through its acetylation in HEK 293 cells. Our data provide strong evidence identifying CBP and p300 as lysine acetyltransferases responsible for the regulation of MR that may provide new therapeutic targets for the treatment of hypertension.

Lipocalin-2 (LCN2) plays an important role in cellular processes as diverse as cell growth, migration/invasion, differentiation, and death/survival. Furthermore, recent studies indicate that LCN2 expression and secretion by glial cells are induced by inflammatory stimuli in the central nervous system. The present study was undertaken to examine the regulation of LCN2 expression in experimental autoimmune encephalomyelitis (EAE) and to determine the role of LCN2 in the disease process. LCN2 expression was found to be strongly increased in spinal cord and secondary lymphoid tissues after EAE induction. In spinal cords astrocytes and microglia were the major cell types expressing LCN2 and its receptor 24p3R, respectively, whereas in spleens, LCN2 and 24p3R were highly expressed in neutrophils and dendritic cells, respectively. Furthermore, disease severity, inflammatory infiltration, demyelination, glial activation, the expression of inflammatory mediators, and the proliferation of MOG-specific T cells were significantly attenuated in Lcn2-deficient mice as compared with wild-type animals. Myelin oligodendrocyte glycoprotein-specific T cells in culture exhibited an increased expression of Il17a, Ifng, Rorc, and Tbet after treatment with recombinant LCN2 protein. Moreover, LCN2-treated glial cells expressed higher levels of proinflammatory cytokines, chemokines, and MMP-9. Adoptive transfer and recombinant LCN2 protein injection experiments suggested that LCN2 expression in spinal cord and peripheral immune organs contributes to EAE development. Taken together, these results imply LCN2 is a critical mediator of autoimmune inflammation and disease development in EAE and suggest that LCN2 be regarded a potential therapeutic target in multiple sclerosis.

Lentiviral short hairpin RNA (shRNA)-mediated genetic screening is a powerful tool for identifying loss-of-function phenotype in mammalian cells. Here, we report the identification of 91 cell migration-regulating genes using unbiased genome-wide functional genetic selection. Individual knockdown or cDNA overexpression of a set of 10 candidates reveals that most of these cell migration determinants are strongly dependent on the PI3K/PTEN/AKT pathway and on their downstream signals, such as FOXO1 and p70S6K1. ALK, one of the cell migration promoting genes, uniquely uses p55 gamma regulatory subunit of PI3K, rather than more common p85 subunit, to trigger the activation of the PI3K-AKT pathway. Our method enables the rapid and cost-effective genome-wide selection of cell migration regulators. Our results emphasize the importance of the PI3K/PTEN/AKT pathway as a point of convergence for multiple regulators of cell migration.

ULK1 (unc-51 like kinase 1) is a serine/threonine protein kinase that plays a key role in regulating the induction of autophagy. Recent studies using autophagy-defective mouse models, such as atg5- or atg7-deficient mice, revealed an important function of autophagy in adipocyte differentiation. Suppression of adipogenesis in autophagy-defective conditions has made it difficult to study the roles of autophagy in metabolism of differentiated adipocytes. In this study, we established autophagy defective-differentiated 3T3-L1 adipocytes, and investigated the roles of Ulk1 and its close homolog Ulk2 in lipid and glucose metabolism using the established adipocytes. Through knockdown approaches, we determined that Ulk1 and Ulk2 are important for basal and MTORC1 inhibition-induced autophagy, basal lipolysis, and mitochondrial respiration. However, unlike other autophagy genes (Atg5, Atg13, Rb1cc1/Fip200, and Becn1) Ulk1 was dispensable for adipogenesis without affecting the expression of CCAAT/enhancer binding protein α (CEBPA) and peroxisome proliferation-activated receptor gamma (PPARG). Ulk1 knockdown reduced fatty acid oxidation and enhanced fatty acid uptake, the metabolic changes that could contribute to adipogenesis, whereas Ulk2 knockdown had opposing effects. We also found that the expression levels of insulin receptor (INSR), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (SLC2A4/GLUT4) were increased in Ulk1-silenced adipocytes, which was accompanied by upregulation of insulin-stimulated glucose uptake. These results suggest that ULK1, albeit its important autophagic role, regulates lipid metabolism and glucose uptake in adipocytes distinctly from other autophagy proteins.

Reactive glia plays a central role in neuroinflammation associated with secondary damage after brain injury. In order to understand the global effects of therapeutic hypothermia on glial activation and neuroinflammation, we performed proteomic profiling of glial cultures following inflammatory stimulation and hypothermic exposure. Primary mixed glial cultures prepared from mouse brains were stimulated with lipopolysaccharide and interferon-γ under normothermic (37°C) or moderate hypothermic (29°C) conditions, and their proteome profiles were compared by LC-ESI-MS/MS. Differentially expressed proteins were determined by high-throughput label-free quantification. Under hypothermic conditions, 64 and 16 proteins were upregulated (≥1.5-fold) and downregulated (≤ 0.7-fold), respectively, compared to normothermic conditions. More importantly, hypothermia altered the abundance of 143 proteins that were either increased or decreased by inflammatory stimulation. The results were validated for several proteins (ICAM-1, STAT-1, YWHAB, and IFIT-3) by Western blot analysis. Pathway and network analysis indicate that hypothermia influences various biological functions of glia such as molecular transport, cell movement, immune response, cell death, and stress response. In conclusion, moderate hypothermia seems to have a significant effect on the protein expression profiles of brain glia and possibly ensuing neuroinflammation. These proteins may be involved in the protective mechanism of hypothermia against brain injuries.

Background Therapeutic hypothermia is one of the neuroprotective strategies that improve neurological outcomes after brain damage in ischemic stroke and traumatic brain injury. Microglial cells become activated following brain injury and play an important role in neuroinflammation and subsequent brain damage. The aim of this study was to determine the time-dependent effects of hypothermia on microglial cell activation and migration, which are accompanied by neuroinflammation. Methods Microglial cells in culture were subjected to mild (33 °C) or moderate (29 °C) hypothermic conditions before, during, or after lipopolysaccharide (LPS) or hypoxic stimulation, and the production of nitric oxide (NO), proinflammatory cytokines, reactive oxygen species, and neurotoxicity was evaluated. Effects of hypothermia on microglial migration were also determined in in vitro as well as in vivo settings. Results Early-, co-, and delayed-hypothermic treatments inhibited microglial production of inflammatory mediators to varying degrees: early treatment was the most efficient, and delayed treatment showed time-dependent effects. Delayed hypothermia also suppressed the mRNA levels of proinflammatory cytokines and iNOS, and attenuated microglial neurotoxicity in microglia-neuron co-cultures. Furthermore, delayed hypothermia reduced microglial migration in the Boyden chamber assay and wound healing assay. In a stab injury model, delayed local hypothermia reduced migration of microglia toward the injury site in the rat brain. Conclusion Taken together, our results indicate that delayed hypothermia is sufficient to attenuate microglial activation and migration, and provide the basis of determining the optimal time window for therapeutic hypothermia. Delayed hypothermia may be neuroprotective by inhibiting microglia-mediated neuroinflammation, indicating the therapeutic potential of post-injury hypothermia for patients with brain damages exhibiting some of the inflammatory components.