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Severe fever with thrombocytopenia syndrome (SFTS) is a new tick-borne viral disease, and most SFTS virus (SFTSV) infections occur via bites from the tick Haemaphysalis longicornis; however, SFTSV transmission can also occur through close contact with an infected patient. SFTS is characterized by acute high fever, thrombocytopenia, leukopenia, elevated serum hepatic enzyme levels, gastrointestinal symptoms, and multiorgan failure and has a 16.2 to 30% mortality rate. In this study, we found that age, dyspnea rates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, multiorgan dysfunction score (MODS), viral load, IL-6 levels, and IL-10 levels were higher in patients with fatal disease than in patients with nonfatal disease during the initial clinical course of SFTS. In addition, we found that IL-6 and IL-10 levels, rather than viral load and neutralizing antibody titers, in patients with an SFTSV infection strongly correlated with outcomes (for severe disease with an ultimate outcome of recovery or death).

To date, hundreds of fungal genomes have been sequenced, and many more are underway. Recently developed cutting-edge techniques generate very large amounts of data, and the field of fungal genomics in dermatology has consequently evolved substantially. Methodological improvements have broadened the scope of large-scale ecological studies in dermatology, including biodiversity assessments and genomic identification of fungi. Here, we aimed to provide a brief introduction to bioinformatic approaches to fungal genomics in the field of dermatology. We described the history and basic concepts of fungal genomics and presented sequencing-based techniques for fungal identification, including a list of the revised taxa of dermatophytes, as determined by current phylogenetic analysis. Finally, we discussed the emerging trends in fungal genomics in dermatology, such as next-generation sequencing.

Serotonin syndrome is an adverse reaction due to increased serotonin (5-hydroxytryptophan: 5-HT) concentrations in the central nervous system (CNS). The full 5-HT1A receptor (5-HT1AR) agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) has been recognized to elicit traditional serotonergic behaviors. Treatment with 8-OH-DPAT selectively increased PKCδ expression out of PKC isoforms and 5-HT turnover rate in the hypothalamus of wild-type mice. Treatment with 8-OH-DPAT resulted in oxidative burdens, co-immunoprecipitation of 5-HT1AR and PKCδ, and phosphorylation and membrane translocation of p47phox. Importantly, p47phox also interacted with 5-HT1AR or PKCδ in the presence of 8-OH-DPAT. Consistently, the interaction and oxidative burdens were attenuated by 5-HT1AR antagonism (i.e., WAY100635), PKCδ inhibition (i.e., rottlerin and genetic depletion of PKCδ), or NADPH oxidase/p47phox inhibition (i.e., apocynin and genetic depletion of p47phox). However, WAY100635, apocynin, or rottlerin did not exhibit any additive effects against the protective effect by inhibition of PKCδ or p47phox. Furthermore, apocynin, rottlerin, or WAY100635 also significantly protected from pro-inflammatory/pro-apoptotic changes induced by 8-OH-DPAT. Therefore, we suggest that 8-OH-DPAT-induced serotonergic behaviors requires oxidative stress, pro-inflammatory, and pro-apoptotic changes, that PKCδ or p47phox mediates the serotonergic behaviors induced by 8-OH-DPAT, and that the inhibition of PKCδ-dependent p47phox activation is critical for protecting against serotonergic behaviors.

Simple Summary The present study shows the role of Drosophila USP14 under ER stress and ER stress related disease, autosomal dominant retinitis pigmentosa. Drosophila USP14 protects cell from ER stress triggered by ER stress-causing chemicals Drosophila S2 cells and suppresses the retinal degeneration in disease model for retinitis pigmentosa by regulating the stability of Rhodopsin-1. This study also indicates the dynamic reorganization of proteasome complex under ER stress. The modulation of USP14 could be a potential therapeutic strategy for treating the diseases associated with protein folding. Abstract Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. Here, we analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, we observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Apical periodontitis is caused by biofilm-mediated root canal infection. Early phase oral bacterial biofilms are inhibited by Lactobacillus plantarum lipoteichoic acid (Lp.LTA). However, mature biofilms that develop over 3 weeks are more resistant to traditional endodontic medicaments. Therefore, this study examined the effectiveness of Lp.LTA on disrupting mature Enterococcus faecalis biofilms, and on enhancing the effects of endodontic medicaments. LTA was purified from L. plantarum through butanol extraction followed by hydrophobic and ion-exchange chromatography. E. faecalis biofilms were formed over 3 weeks on glass bottom dishes and in dentin blocks obtained from human single-rooted premolars. These mature biofilms were treated with or without Lp.LTA for 1 h, followed by additional treatment with either chlorhexidine digluconate (CHX), calcium hydroxide (CH), or triple antibiotics for 24 h. Biofilms on glass were live/dead stained and quantified by ZEN through confocal laser microscopy. Bio-films in dentin were fixed, sputter coated and analyzed by ImageJ with scanning electron microscopy. Preformed E. faecalis mature biofilms on the culture dishes were dose-dependently disrupted by Lp.LTA. Lp.LTA potentiated the effects of CHX or CH on the disruption of mature biofilm. Interestingly, CHX-induced disruption of preformed E. faecalis mature biofilms was synergistically enhanced only when pre-treated with Lp.LTA. Furthermore, in the dentin block model, Lp.LTA alone reduced E. faecalis mature biofilm and pre-treatment with Lp.LTA promoted the anti-biofilm activity of CHX. Lp.LTA could be an anti-biofilm or supplementary agent that can be effective for E. faecalis-biofilm-induced diseases.

A previous study of ours indicated that Drosophila flightless-1 controls lipid metabolism, and that there is an accumulation of triglycerides in flightless-1 (fliI)-mutant flies, where this mutation triggers metabolic stress and an obesity phenotype. Here, with the aim of characterizing the function of FliI in metabolism, we analyzed the levels of gene expression and metabolites in fliI-mutant flies. The levels of enzymes related to glycolysis, lipogenesis, and the pentose phosphate pathway increased in fliI mutants; this result is consistent with the levels of metabolites corresponding to a metabolic pathway. Moreover, high-throughput RNA sequencing revealed that Drosophila FliI regulates the expression of genes related to biological processes such as chromosome organization, carbohydrate metabolism, and immune reactions. These results showed that Drosophila FliI regulates the expression of metabolic genes, and that dysregulation of the transcription controlled by FliI gives rise to metabolic stress and problems in the development and physiology of Drosophila. [BMB Reports 2018; 51(9): 462-467].

Summary: The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell. : Kim et al. present evidence that the ubiquitin-proteasome system and autophagy are in a compensatory negative-feedback connection through USP14, a proteasome-associated deubiquitinating enzyme. USP14 inhibition results in elevation of proteasome activity and facilitation of tau degradation in the cell, while it delays the cellular autophagic flux. Keywords: USP14, proteasome, ubiquitin-proteasome system, autophagy, autophagic flux, negative feedback, UVRAG, proteostasis, tau, huntingtin

Background/Aims: The 26S proteasome is the key proteolytic complex for recognition and degradation of polyubiquitinated target substrates in eukaryotes. Among numerous proteasome-associated proteins, a deubiquitinating enzyme (DUB) USP14 has been identified as an endogenous inhibitor of the proteasome. Here, we explored the complex regulatory functions of USP14 that involve ubiquitin (Ub) homeostasis and substrate degradation in flies and mammals. Methods: USP14-null primary and immortalized mouse embryonic fibroblasts (MEFs) and USP14 knocked-down Drosophila were analyzed in this study. We measured proteasome and DUB activities using fluorogenic reporter substrates and adduct-forming probes. To examine the levels of ubiquitin, we performed immunoblotting and immunohistochemistry. Mass spectrometry (MS) was used to examine polyUb chain linkages and USP14-interacing proteins. Cell cycle was analyzed by flow cytometry, BrdU labeling, and phospho-histone H3 staining. Results: The homeostasis of Ub in USP14–/–MEFs was markedly perturbed because of facilitated clearance of Ub. This phenomenon was recapitulated in muscles of USP14-deficient Drosophila with old ages. Absolute quantitation using MS also revealed that USP14–/– MEFs contained significantly increased amounts of Ub, compared with wild-type. The key phenotype of USP14–/– MEFs was their delayed proliferation originated from prolonged interphase possibly through aberrant degradation of cyclins A and B1. We found that knocking down USP14 in Drosophila resulted in delayed eye development associated with reduced mitotic activity. Conclusion: Our study identifies novel cellular functions of USP14 not only in cellular Ub hometostasis but also in cell cycle progression. USP14 was also essential for proper Drosophila eye development. These results strongly suggest that the USP14-mediated proteasome activity regulation may be directly related to various human diseases including cancer.

// Jung Nyeo Chun 1, 2, * , Soonbum Park 1, * , Sanghoon Lee 3 , Jae-Kyung Kim 1 , Eun-Jung Park 1 , MinJi Kang 1 , Hye Kyung Kim 4, 5 , Jong Kwan Park 5 , Insuk So 1, 2 and Ju-Hong Jeon 1, 2 1 Department of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Korea 2 Institute of Human-Environment Interface Biology, Seoul National University, Seoul 03080, Korea 3 Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112-5650, USA 4 College of Pharmacy, Kyungsung University, Busan 48434, Korea 5 Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, Jeonju 54896, Korea * These authors contributed equally to this work Correspondence to: Ju-Hong Jeon, email: jhjeon2@snu.ac.kr Keywords: schisandra chinensis; schisandrol B; schisandrin B; TGFβ1; NF-κB Received: June 30, 2017 Accepted: November 15, 2017 Published: December 14, 2017 ABSTRACT Aberrant transforming growth factor β1 (TGFβ1) signaling plays a pathogenic role in the development of vascular fibrosis. We have reported that Schisandra chinensis fruit extract (SCE), which has been used as a traditional oriental medicine, suppresses TGFβ1-mediated phenotypes in vascular smooth muscle cells (VSMCs). However, it is still largely unknown about the pharmacologic effects of SCE on various TGFβ1 signaling components. In this study, we found that SCE attenuated TGFβ1-induced NF-κB activation and nuclear translocation in VSMCs. Among the five active ingredients of SCE that were examined, schisandrol B (SolB) and schisandrin B (SchB) most potently suppressed TGFβ1-mediated NF-κB activation. In addition, SolB and SchB effectively inhibited IKKα/β activation and IκBα phosphorylation in TGFβ1-treated VSMCs. The pharmacologic effects of SolB and SchB on NF-κB activation were independent of the Smad-mediated canonical pathway. Therefore, our study demonstrates that SCE and its active constituents SolB and SchB suppress TGFβ1-mediated NF-κB signaling pathway in a Smad-independent mechanism. Our results may help further investigations to develop novel multi-targeted therapeutic strategies that treat or prevent vascular fibrotic diseases.

Geraniol, an acyclic dietary monoterpene, has been found to suppress cancer survival and growth. However, the molecular mechanism underlying the antitumor action of geraniol has not been investigated at the genome‐wide level. In this study, we analyzed the microarray data obtained from geraniol‐treated prostate cancer cells. Geraniol potently altered a gene expression profile and primarily down‐regulated cell cycle‐related gene signatures, compared to linalool, another structurally similar monoterpene that induces no apparent phenotypic changes. Master regulator analysis using the prostate cancer‐specific regulatory interactome identified that the transcription factor E2F8 as a specific target molecule regulates geraniol‐specific cell cycle signatures. Subsequent experiments confirmed that geraniol down‐regulated E2F8 expression and the knockdown of E2F8 was sufficient to suppress cell growth by inducing G2/M arrest. Epidemiological analysis showed that E2F8 is up‐regulated in metastatic prostate cancer and associated with poor prognosis. These results indicate that E2F8 is a crucial transcription regulator controlling cell cycle and survival in prostate cancer cells. Therefore, our study provides insight into the role of E2F8 in prostate cancer biology and therapeutics.

The number of octapeptide repeats has been considered to correlate with clinical and pathogenic phenotypes of prion diseases resulting from aberrant metabolism of prion protein (PrP). However, it is still poorly understood how this motif affects PrP metabolism. Here, we discover homozygous single octapeptide repeat deletion mutation in the PRNP gene encoding PrP in HeLa cells. The level of PrP proves to be unaffected by this mutation alone, but selectively reduced by additional pathogenic mutations within internal hydrophobic region of PrP. The pattern and relative amount of newly synthesized A117V mutant is unaffected, whereas the mutant appears to be differentially distributed and processed on the cell surface by single octapeptide deletion. This study provides an insight into a novel mutant-specific metabolism of PrP on the cell surface.

To report long-term outcomes of intravitreal ceftazidime injection in patients with endogenous Klebsiella pneumoniae endophthalmitis (EKPE).This was a retrospective observational case study, including 7 eyes from 6 patients with EKPE. The medical records from January 2010 to December 2018 were reviewed.Diagnosis of EKPE was made based on the finding of endophthalmitis with concurrent systemic infection and positive blood culture result. All patients received tap and intravitreal ceftazidime injection base on the results of antibiotics sensitivity test. Visual acuity ranged from no light perception to 20/60 at initial visit, and the final visual acuity was 20/20. Two eyes underwent evisceration after intravitreal injection.Intravitreal ceftazidime injection showed favorable results in patients with EKPE.

Neutrophils may be involved in the local pathophysiology of chronic graft-versus-host disease (cGVHD). We evaluated neutrophil infiltration in cGVHD using conjunctival impression cytology (IC) and its clinical correlation with ocular surface status and neutrophil enzyme levels in tears.This cross-sectional observational study included 76 patients with cGVHD. The ocular surface was assessed for the tear break-up time, Schirmer I test, corneal and conjunctival staining score, meiboscore, and the ocular surface disease index questionnaire. Conjunctival IC was performed at the temporal, superior bulbar, and upper palpebral conjunctiva, and the number of neutrophils (cells/high power field [HPF]) was calculated. Neutrophil elastase (NE), myeloperoxidase (MPO), and matrix metalloperoxidase-8 and -9 levels in tear washes were measured in 20 patients.The number of neutrophils was significantly greater at the upper palpebral conjunctiva (median [range], 16.5 [0 to 147] cells/HPF) than at the temporal and superior bulbar conjunctiva (0 [0 to 70] and 0 [0 to 105] cells/HPF; P 0.0001). The number of neutrophils at the upper palpebral conjunctiva showed moderate correlations with the corneal staining score and the NE and MPO levels in tears (r = 0.668, 0.553, and 0.563, respectively; P 0.0001, P = 0.014, and 0.012).Our results suggest that neutrophils at the upper palpebral conjunctiva associate with the clinical manifestations and inflammatory status of the ocular surface in cGVHD. Conjunctival neutrophils should be addressed when assessing the inflammatory activity of ocular cGVHD and exploring its pathogenesis.

Background: Although Candida albicans is considered to be the major fungal pathogen of candidemia, severe infections by non-albicans Candida (NAC) spp. have been on the increase in recent years. Among NAC spp., C. glabrata has emerged as the second most common pathogen. Unlike other Candida spp., it is often resistant to various azole antifungal agents, such as fluconazole. However, few studies have been conducted to investigate its structure, epidemiology, and basic biology. Recently, multi-locus sequence typing (MLST) has been developed as a highly useful and portable molecular biology technique. Methods: In the present study, MLST was performed with a total of 102 C. glabrata clinical isolates that were isolated from various types of clinical specimens. The present study was performed with a total of 102 C. glabrata clinical isolates that were isolated from various types of clinical specimens. The fungal internal transcribed spacer (ITS) gene wad amplified and sequenced to identify and confirm C. glabrata clinical isolates. For MLST, six housekeeping genes including 1,3-beta-glucan synthase (FKS), 3-isopropylmalate dehydrogenase (LEU2),myristoyl-CoA, protein Nmyristoyltransferase (NMT1), phosphoribosyl-anthranilate isomerase (TRP1), UTP-glucose-1-phosphate uridylyltransferase (UGP1), and orotidine-5'-phosphate decarboxylase (URA3) were amplified and sequenced. The results were analyzed by using the C. glabrata database. Results: Of a total of 3,345 base-pair DNA sequences, 49 (1.5%) variable nucleotide sites were found and the results showed that a total of 12 different sequence types (STs) were identified from the 102 clinical isolates. As classified by STs, The ST138 was the most predominant sequence type (ST) in this study as a result of 52.9% (54/102), and the following most predominant ST was the ST63 as a result of 23.5% (24/102). Conclusion: In conclusion, this data demonstrated that the ST138 was the most predominant ST in Korea. Further, we found eight undetermined STs (USTs) and then seven STs among these STs were given the number by PubMLST database. The data from this study might provide a fundamental database for further studies on C. glabrata, including its epidemiology, and evolution. Furthermore, the data might also contribute to the development of novel antifungal agents and diagnostic tests.

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser1412) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune-modulatory receptor involved in phagocytosis and inflammation. Mutations of Q33X, Y38C and T66M cause Nasu-Hakola disease (NHD) which is characterized by early onset of dementia and bone cysts. A recent, genome-wide association study also revealed that single nucleotide polymorphism of TREM2, such as R47H, increased the risk of Alzheimer's disease (AD) similar to ApoE4. However, how these mutations affect the trafficking of TREM2, which may affect the normal functions of TREM2, was not known. In this study, we show that TREM2 with NHD mutations are impaired in the glycosylation with complex oligosaccharides in the Golgi apparatus, in the trafficking to plasma membrane and further processing by γ-secretase. Although R47H mutation in AD affected the glycosylation and normal trafficking of TREM2 less, the detailed pattern of glycosylated TREM2 differs from that of the wild type, thus suggesting that precise regulation of TREM2 glycosylation is impaired when arginine at 47 is mutated to histidine. Our results suggest that the impaired glycosylation and trafficking of TREM2 from endoplasmic reticulum/Golgi to plasma membrane by mutations may inhibit its normal functions in the plasma membrane, which may contribute to the disease.

Atrazine (ATR) is one of the most commonly applied broad-spectrum herbicides. Although ATR is well known to be a biologically hazardous molecule with potential toxicity in the immune system, the molecular mechanisms responsible for ATR-induced immunotoxicity remain unclear. In this study, we found that the immunotoxic properties of ATR were mediated through the induction of apoptotic changes in T lymphocytes. Mice exposed to ATR for 4 weeks exhibited a significant decrease in the number of spleen CD3(+) T lymphocytes, while CD19(+) B lymphocytes and nonlymphoid cells were unaffected. ATR exposure also led to inhibition of cell growth and induction of apoptosis in human Jurkat T-cells. Importantly, ATR triggered the activation of caspase-3 and the cleavage of caspase-8 and PARP, whereas it did not affect the release of cytochrome c from the mitochondria in Jurkat T-cells. In addition, ATR activated the unfolded protein response signaling pathway, as indicated by eIF2α phosphorylation and CHOP induction. Our results demonstrate that ATR elicited an immunotoxic effect by inducing ER stress-induced apoptosis in T-cells, therefore providing evidence for the molecular mechanism by which ATR induces dysregulation of the immune system. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 998-1008, 2016.

Macroautophagy (autophagy) is believed to maintain energy homeostasis by degrading unnecessary cellular components and molecules. Its implication in regulating cancer metabolism recently started to be uncovered. However, the precise roles of autophagy in cancer metabolism are still unclear. Here, we show that autophagy plays a critical role in glutamine metabolism, which is required for tumor survival. Pancreatic ductal adenocarcinoma (PDAC) cells require both autophagy and typical glutamine transporters to maintain intracellular glutamine levels. Glutamine deprivation, but not that of glucose, led to the activation of macropinocytosis-associated autophagy through TFEB induction and translocation into the nucleus. In contrast, glutamine uptake increased as a compensatory response to decreased intracellular glutamine levels upon autophagy inhibition. Moreover, autophagy inhibition and glutamine deprivation did not induce cell death, while glutamine deprivation dramatically activated apoptotic cell death upon autophagy inhibition. Interestingly, the addition of α-ketoglutarate significantly rescued the apoptotic cell death caused by the combination of the inhibition of autophagy with glutamine deprivation. Our data suggest that macropinocytosis-associated autophagy is a critical process providing glutamine for anaplerosis of the TCA cycle in PDAC. Thus, targeting both autophagy and glutamine metabolism to completely block glutamine supply may provide new therapeutic approaches to treat refractory tumors.

Chronic stress in the endoplasmic reticulum (ER) underlies many degenerative and metabolic diseases involving apoptosis of vital cells. A well-established example is Autosomal Dominant Retinitis Pigmentosa (ADRP), an age-related retinal degenerative disease caused by mutant rhodopsins 1, 2. Similar mutant alleles of Drosophila rhodopsin-1 also impose stress on the ER and cause age-related retinal degeneration in that organism 3. Well-characterized signaling responses to ER-stress, referred to as the Unfolded Protein Response (UPR) 4, induce various ER quality control genes that can suppress such retinal degeneration 5. However, how cells activate cell death programs after chronic ER-stress remains poorly understood. Here, we report the identification of a signaling pathway mediated by cdk5 and mekk1 required for ER-stress-induced apoptosis. Inactivation of these genes specifically suppressed apoptosis, without affecting other protective branches of the UPR. Cdk5 phosphorylates Mekk1, and together, activate the JNK pathway for apoptosis. Moreover, disruption of this pathway can delay the course of age-related retinal degeneration in a Drosophila model of ADRP. These findings establish a previously unrecognized branch of ER-stress response signaling involved in degenerative diseases.

Mutations in the rhodopsin gene that disrupt the encoded protein's folding properties are a major cause of autosomal dominant retinitis pigmentosa (ADRP). This disease is faithfully modeled in Drosophila where similar mutations in the ninaE gene, encoding rhodopsin-1 (Rh-1), cause ER stress and dominantly trigger age-related retinal degeneration. In addition, mutant flies bearing certain ninaE alleles have dramatically reduced Rh-1 protein levels, but the underlying mechanism for this reduction and significance of its contribution to the ADRP phenotype remains unclear. To address this question, we specifically analyzed the role of Drosophila genes homologous to the known yeast and animal regulators of the ER-associated degradation (ERAD) pathway, a process that reduces levels of misfolded proteins in the ER through proteasomal degradation. We found that loss-of-function of these putative ERAD factors resulted in increased levels of Rh-1 in ninaE mutant flies. Conversely, in an ER stress assay where mutant or wild-type Rh-1 were overexpressed in developing imaginal discs beyond the ER protein folding capacity of those cells, co-expression of certain ERAD factors was sufficient to reduce Rh-1 protein levels and to completely suppress ER stress reporter activation. Significantly, those ERAD factors that specifically reduced misfolded Rh-1 in the imaginal disc assay also delayed age-related retinal degeneration caused by an endogenous ninaE allele, indicating that ERAD acts as a protective mechanism against retinal degeneration in the Drosophila model for ADRP. These results suggest that manipulation of ERAD may serve as a powerful therapeutic strategy against a number of diseases associated with ER stress.

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. Here, we analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaE(G69D)-/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models.

The responses to ionizing radiation (IR) in tumors are dependent on cellular context. We investigated radiation-related expression patterns in Jurkat T cells with nonsense mutation in p53 using cDNA microarray. Expression of 2400 genes in gamma-irradiated cells was distinct from other stimulations like anti-CD3, phetohemagglutinin (PHA) and concanavalin A (ConA) in unsupervised clustering analysis. Among them, 384 genes were selected for their IR-specific changes to make 'RadChip'. In spite of p53 status, every type of cells showed similar patterns in expression of these genes upon gamma-radiation. Moreover, radiation-induced responses were clearly separated from the responses to other genotoxic stress like UV radiation, cisplatin and doxorubicin. We focused on two IR-related genes, phospholipase Cgamma2 (PLCG2) and cytosolic epoxide hydrolase (EPHX2), which were increased at 12 h after gamma-radiation in RT-PCR. TPCK could suppress the induction of these two genes in either of Jurkat T cells and PBMCs, which might suggest the transcriptional regulation of PLCG2 and EPHX2 by NF-kappaB upon gamma-radiation. From these results, we could identify the IR-specific genes from expression profiling, which can be used as radiation biomarkers to screen radiation exposure as well as probing the mechanism of cellular responses to ionizing radiation.

SummaryThe mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. Here, we report that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. We identified cdk7 through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppressed apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid failed to localize to the mitochondria and failed to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid.

Microalgae have recently been recognized as a valuable resource for various applications. Phaeodactylum tricornutum is a diatom that lives in marine water and has an unusually high content of lipids. In this study, we added P. tricornutum into a gelatin dope solution to examine the effect of this diatom using electrospinning. The addition of P. tricornutum extracts increased the conductivity of the dope solution but had little effect on the viscosity. Due to the increased conductivity, the fiber diameter was reduced compared with the neat gelatin nanofiber. The loading of P. tricornutum extracts was confirmed using fluorescence microscopy, and the incorporation of lipids was detected through gas chromatography. The P. tricornutum-loaded nanofiber mat exhibited anti-microbial activity against Escherichia coli and multidrug-resistant Staphylococcus aureus (MRSA). The cell viability test showed that the P. tricornutum-loaded nanofiber has no cytotoxicity. We expect that this antimicrobial P. tricornutum-loaded gelatin nanofiber mat can be applied as a wound dressing.

Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. Here, we report that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, we developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1p>dsRed transgenic flies showed patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression was highest in salivary glands and the intestine. In the adult, the male reproductive organs showed high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1p>dsRed reporter was induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells.

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 x Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.

Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5’-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5’ UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.

Growth factors such as basic fibroblast growth factors (bFGFs) could induce the differentiation of mouse neuroblastoma cells. We examine the effect of caveolin-1 on bFGF-induced differentiation of N2a cells. Caveolin-1 blocked the formation of neurites and the phosphorylation of Erk upon bFGF treatment in N2a cells. Active mutants of Rho family small GTPases (Rac1 and Cdc42) could not affect the inhibitory effect of caveolin-1, but we could restore the differentiation of N2a cells by introducing active mutants of p21-activated kinase 1 (PAK1). Over-expressed caveolin-1 could be coimmunoprecipitated with PAK1, which interrupted the steady-state Rac1/Cdc42-PAK1 interactions. From these results, we suggest that the up-regulated caveolin-1 in neuronal cells can inhibit the bFGF signaling pathway from small GTPases to PAK1 by directly binding to PAK1.

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, beta APP secretion was up-regulated by caveolin-1 over- expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by beta-secretase via PKC downregulation.

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.