Cellular Catabolic Process Research Articles

Inhibiting the Core Autophagy Enzyme ATG4B with Novel Drugs Sensitizes Resistant Leukemic Stem/Progenitor Cells to Standard Targeted Therapy

Leukemic stem cell (LSC) persistence is a major cause of therapy failure and relapse in patients, thus warranting further investigations into the cells' molecular properties to aid in meaningful clinical decisions. Chronic myeloid leukemia (CML) is a clear example and excellent study model with rare, propagating LSCs that are not eradicated by BCR-ABL1-directed tyrosine kinase inhibitor (TKI) monotherapy such as Imatinib Mesylate (IM), since the LSCs do not exclusively rely on BCR-ABL1 for their survival.We and others have shown that the persistence of primitive leukemic cells is mediated by macroautophagy (autophagy), a catabolic cellular recycling process. In particular, we discovered that transcript and protein expressions of all ATG4 family members, including ATG4A, B, C, and D, are significantly increased in CD34+ CML cells compared to CD34+ normal bone marrow (BM) cells. ATG4B expression is also significantly higher in pre-treatment CD34+ CML cells from IM-nonresponders vs. IM-responders, including LSCs. Moreover, we revealed that stable suppression of ATG4B significantly suppressed autophagy, impaired survival of IM-nonresponder stem/progenitor cells and sensitized leukemic cells to TKI treatment. Thus, we identified ATG4B as a critical therapeutic target in CML (Rothe et al., Blood, 2014).To further explore whether targeting of ATG4B would be a viable strategy in the successful treatment of various leukemia, two lead ATG4B inhibitors were recently developed for pre-clinical proof-of-concept studies. Compound 4-28, a styrylquinoline, was identified by in silico screening and high content cell-based screening. Its structure-activity relationship (SAR)-based optimization led to a more stable and potent compound, LV-320. LV-320 was further evaluated by Microscale Thermophoresis and showed consistent Kd values for binding to the ATG4B enzyme (Kd=16±1 μM). LV-320 can inhibit autophagic flux, shows excellent tolerability and a good PK profile. It is also characterised as a non-competitive inhibitor of ATG4B and displays similar potency against ATG4A. Interestingly, inhibition of ATG4B with compound 4-28 decreased viability by 40-60% and increased apoptosis by 30-40% in different BCR-ABL1+ leukemic cell lines upon serum-deprivation as compared to the same cells without treatment of 4-28 (p<0.05). 4-28 treatment also efficiently inhibited autophagic flux in these cells as shown by Western blot analysis of LC3-II/I and p62 accumulation. In addition, compound 4-28 was able to inhibit the clonal growth of patient-derived CML stem/progenitor cells from IM-nonresponders, in particular when combined with various TKIs compared to single agents (20 vs. 50%, p<0.05). However, this combination approach also showed slight toxic effects on healthy BM cells.We then tested the more stable and potent compound LV-320 with superior results. Treatment of several drug-resistant and mutated CML and aggressive BCR-ABL1+ B-ALL cell lines with LV-320 increased apoptosis up to 90% compared to controls and reached almost 100% when combined with various TKIs (p<0.05). Moreover, LV-320 sensitized IM-nonresponder stem and progenitor cells to TKIs in colony-forming short-term and long-term cell assays as compared to single agents (10 % vs. 40 %, p<0.05), but was not toxic to primitive BM cells from healthy donors (up to 10µM). Mechanistically, we found that LV-320 effectively inhibited autophagic flux in leukemic cells: Confocal microscopy demonstrated an increase in LC3-II-positive punctae (autophagosomes) and the presence of yellow punctae (blocked autophagosome-lysosome fusion) when leukemic cells where transduced with a mRFP-GFP-LC3 construct and treated with LV-320 or a combination of LV-320 and TKIs. Drug interaction analysis further indicated synergy between LV-320 plus IM (CI value ≤ 0.9). A novel in vivo model is currently being investigated to validate our proof-of-concept. Together, our results suggest that targeting of ATG4B with novel autophagy inhibitors in combination with TKIs may be able to circumvent drug resistance in CML and possibly other aggressive leukemia. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide transcriptional response of microRNAs to the benzo(a)pyrene stress in amphioxus Branchiostoma belcheri

Amphioxus, a cephalochordate found in sand habitats in shallow in-shore seawaters, has been widely used as a model in comparative immunology of chordates. However, the role of microRNAs (miRNAs) in amphioxus under abiotic stress, particularly xenobiotics with strong toxicity, remains largely unknown. Here, a widespread marine contaminant, benzo(a)pyrene (BaP) is used to evaluate its toxic effects on miRNA expression of amphioxus. Six small RNA libraries were sequenced from Branchiostoma belcheri. A total of 144 known and 157 novel miRNAs were identified using deep sequencing and bioinformatics approaches. A total of 58 differentially expressed miRNAs (DEMs) were screened, including 25 up- and 33 down-regulated DEMs under BaP stress. Target genes possibly regulated by DEMs were predicted, and their functional enrichment analyses were performed. Targets of DEMs are primarily involved in xenobiotic and cellular homeostasis, catabolic and transport process. They could be largely linked to nine immune- and toxin detoxification-related pathways, including metabolism of xenobiotics by cytochrome P450, drug metabolism-other enzymes, and drug metabolism-cytochrome P450, etc. Furthermore, quantitative real-time PCR (qRT-PCR) analysis for 12 key BaP-responsive DEMs validates the accuracy of deep sequencing. Experiments were then conducted to investigate their expression responses to BaP stress at different time intervals in detail to further determine their expression dynamic in responses of B. belcheri towards BaP exposure. This study, to the best of our knowledge, investigates the regulatory roles of miRNAs in the toxicological response of amphioxus for the first time, providing valuable information for the protection of lone existing cephalochordate amphioxus.

Gene correlation network analysis to identify regulatory factors in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a severe lung disease characterised by extensive pathological changes. The objective for this study was to identify the gene network and regulators underlying disease pathology...

Hepatic transcript profiling in early-lactation dairy cows fed rumen-protected niacin during the transition from late pregnancy to lactation

In dairy cows, administration of high dosages of niacin (nicotinic acid, NA) was found to cause antilipolytic effects, which are mediated by the NA receptor hydroxyl-carboxylic acid receptor 2 (HCAR2) in white adipose tissue (WAT), and thereby an altered hepatic lipid metabolism. However, almost no attention has been paid to possible direct effects of NA in cattle liver, despite evidence that HCAR2 is also expressed in the liver and is even more abundant than in WAT. Because of this, we hypothesized that feeding a high dosage of rumen-protected NA to dairy cows influences critical metabolic or signaling pathways in the liver by inducing changes in the hepatic transcriptome. To identify these pathways, we applied genome-wide transcript profiling in liver biopsies obtained at d 7 postpartum (p.p.) from dairy cows used in our recent study; cows received either no NA (control group, n = 9) or 79 mg of rumen-protected NA/kg of body weight daily (NA group, n = 9) from 21 d before calving until 3 wk p.p. Hepatic transcript profiling revealed that 487 transcripts were differentially expressed (filter criteria: fold change >1.2 or <-1.2 and P < 0.05) in the liver at d 7 p.p. between cows fed NA and control cows. Substantially more transcripts were downregulated (n = 338), whereas only 149 transcripts were upregulated by NA in the liver of cows. Gene set enrichment analysis for the upregulated transcripts revealed that the most-enriched gene ontology biological process terms were exclusively related to immune processes, such as leukocyte differentiation, immune system process, activation of immune response, and acute inflammatory response. Gene set enrichment analysis of the downregulated transcripts showed that the most-enriched biological process terms were related to metabolic processes, such as cellular metabolic process, small molecule metabolic process, lipid catabolic process, organic cyclic compound metabolic process, small molecule biosynthetic process, and cellular lipid catabolic process. In conclusion, hepatic transcriptome analysis showed that rumen-protected NA induces genes that are involved mainly in immune processes, including acute phase response and stress response, in dairy cows at d 7 p.p. Thus, supplementation of a high dosage of rumen-protected NA to dairy cows in the periparturient period may induce or amplify the systemic inflammation-like condition that is typically observed in the liver of high-yielding dairy cows in the p.p. period.

The Role of AMPK in the Regulation of Skeletal Muscle Size, Hypertrophy, and Regeneration.

AMPK (5’-adenosine monophosphate-activated protein kinase) is heavily involved in skeletal muscle metabolic control through its regulation of many downstream targets. Because of their effects on anabolic and catabolic cellular processes, AMPK plays an important role in the control of skeletal muscle development and growth. In this review, the effects of AMPK signaling, and those of its upstream activator, liver kinase B1 (LKB1), on skeletal muscle growth and atrophy are reviewed. The effect of AMPK activity on satellite cell-mediated muscle growth and regeneration after injury is also reviewed. Together, the current data indicate that AMPK does play an important role in regulating muscle mass and regeneration, with AMPKα1 playing a prominent role in stimulating anabolism and in regulating satellite cell dynamics during regeneration, and AMPKα2 playing a potentially more important role in regulating muscle degradation during atrophy.

Defective autophagy in vascular smooth muscle cells enhances atherosclerotic plaque instability

Autophagy is considered as an evolutionarily conserved cellular catabolic process. Defective autophagy has been implicated in various human diseases, including cardiovascular diseases. Recently, we and others demonstrated that defective autophagy in vascular smooth muscle cells (SMCs) promotes the progression of atherosclerosis. In this study, we investigated the role of autophagy in SMCs on plaque instability in vivo. We generated mice with a defect atg7in which is an essential gene for autophagy, in SMCs by crossing Atg7f/f mice with transgelin (Tagln) Cre+/0 mice (Atg7cKO). Then, Atg7cKO and apolipoprotein E (apoe)-deficient (apoeKO) mice were crossed to generate Atg7cKO:apoeKO mice. To generate a mouse model of plaque instability, we conducted to form a tandem stenosis in the carotid artery of Atg7cKO:apoeKO mice and their controls (apoeKO mice) at the age of 10 weeks. At 5 weeks after surgery, the percentage of cross-sectional stenosis area in the operated common carotid artery of Atg7cKO:apoeKO mice was significantly higher than that in apoeKO mice. In addition, thrombus, which was not observed in apoeKO mice, was frequently found in Atg7cKO:apoeKO mice. Furthermore, the number of Berlin blue staining-positive areas, which indicated intraplaque hemorrhage, was significantly higher in Atg7cKO:apoeKO mice than in control apoeKO mice. Taken together, our data suggest that defective autophagy in SMCs enhances plaque instability and the risk of plaque rupture.

Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1

ABSTRACTMicroglia are innate immune cells in the central nervous system (CNS), that supplies neurons with key factors for executing autophagosomal/lysosomal functions. Macroautophagy/autophagy is a cellular catabolic process that maintains cell balance in response to stress-related stimulation. Abnormal autophagy occurs with many pathologies, such as cancer, and autoimmune and neurodegenerative diseases. Hence, clarification of the mechanisms of autophagy regulation is of utmost importance. Recently, researchers presented microRNAs (miRNAs) as novel and potent modulators of autophagic activity. Here, we found that Mir223 deficiency significantly ameliorated CNS inflammation, demyelination and the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and increased resting microglia and autophagy in brain microglial cells. In contrast, the autophagy inhibitor 3-methylademine (3-MA) aggravated the clinical symptoms of EAE in wild-type (WT) and Mir223-deficienct mice. Furthermore, it was confirmed that Mir223 deficiency in mice increased the protein expression of ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and LC3-II in bone marrow-derived macrophage cells compared with cells from WT mice. Indeed, the cellular level of Atg16l1 was decreased in BV2 cells upon Mir223 overexpression and increased following the introduction of antagomirs. We also showed that the 3’ UTR of Atg16l1 contained functional Mir223-responsive sequences and that overexpression of ATG16L1 returned autophagy to normal levels even in the presence of Mir223 mimics. Collectively, these data indicate that Mir223 is a novel and important regulator of autophagy and that Atg16l1 is a Mir223 target in this process, which may have implications for improving our understanding of the neuroinflammatory process of EAE.Abbreviations: 3-MA: 3-methylademine; ACTB/β-actin: actin, beta; ATG: autophagy related; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CNR2: cannabinoid receptor 2 (macrophage); CNS: central nervous system; CQ: chloroquine; EAE: experimental autoimmune encephalomyelitis; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H&E: hematoxylin and eosin; ITGAM: integrin alpha M; LPS: lipoplysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; miRNAs: microRNAs; MS: multiple sclerosis; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; RA: rheumatoid arthritis; SQSTM1: sequestosome 1; TB: tuberculosis; TIMM23: translocase of inner mitochondrial membrane 23; TLR: toll-like receptor.

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

ABSTRACT Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.

Genetic ablation of adenosine receptor A3 results in articular cartilage degeneration.

Adenosine receptor A3 (A3) knockout results in progressive loss of articular cartilage in vivo. Ablation of A3 results in activation of matrix degradation and cartilage hypertrophy. A3agonists downregulate RUNX2 and CaMKII expression in osteoarthritic human articular chondrocytes. A3 prevents articular cartilage matrix degradation induced by inflammation and osmotic fluctuations. A3 agonist inhibits proteolytic activity of cartilage-degrading enzymes.

Abstract 5271: Exercise intervention alleviates inflammation-related biomarkers in breast cancer survivors

Abstract Purpose: In this pilot study we assessed the feasibility of breast cancer survivors exercising at Curves facilities, and examined stress-related biomarkers to explain the association between exercise and reduction in cancer associated co-morbidities. Methods: 50 sedentary, estrogen-positive, postmenopausal breast cancer survivors were recruited from the City of Hope, of whom 50% were randomized into a 16-week Curves exercise intervention. Adherence to the exercise/control regimen was followed through the 16 weeks. Body measurements and blood samples were taken at each time point. Samples were analyzed using genome-wide RNA sequencing and inflammation measured using a 13 Plex Immunology Multiplex Assay and a C-reactive protein assay. Results: Although insignificant variation in weight loss was observed between the two groups after 16 weeks, the exercise group did show a reduction in 12 of the 14 measurable inflammation markers compared to the control group. The average fold change after 16 weeks showed that women in the exercise group had a 40% and 25% reduction in the proinflammatory markers CRP and IL-6, respectively, when compared to the control group. Additionally, a greater number of women in the exercise group showed an overall decrease in proinflammatory markers including IL-6, CRP, TNF-α and IL-8 when compared to the control group; 80% of the women in the exercise group showed reduced levels of IL-8 at 16 weeks compared to control group where only 36.4% of women had any reduction in IL-8 levels. Interestingly, irrespective of the exercise intervention, analyses based on their exposure to radiation therapy (RT), showed that women who underwent RT exhibited increased levels of all 14 inflammation markers at 16-weeks while women who had not been given RT showed a slight decrease in 13 of the 14 inflammation markers. The gene expression profiling results for the exercise group showed 435 differentially expressed (DE) genes post-intervention, when compared to the gene expression at baseline (p=0.05). Several of these DE genes are involved with inflammatory response, interleukin and interferon signaling pathways. Interestingly, the difference at baseline between the breast cancer survivors from this study and a group of women without a history of cancer from a parallel exercise study by our team showed a drastic variation, with a total of 2,543 DE genes (p=0.05), with most of the DE genes associated with catabolic cellular processes. Conclusion: Our results indicate that moderate levels of exercise, even without weight loss, could potentially be useful in alleviating inflammation and stress-related biomarkers in breast cancer survivors. The expression-level variation at baseline between women with and without a history of breast cancer could be attributed to the oncogenic and chemotherapy-related stress in the breast cancer survivors. Further gene set enrichment analysis is needed for all groups. Citation Format: Aditi Vyas, Yuan Chun Ding, Sarah Flores, Megan Johnson, Gabrielle Riazi, Joanne Mortimer, Susan Neuhausen, Jessica Clague Dehart. Exercise intervention alleviates inflammation-related biomarkers in breast cancer survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5271.

Targeting autophagy in liver cancer.

Autophagy is a catabolic cellular process conserved in animals. It is characterized by the main role of recycling all the non-functional products of the cells. Once, autophagy players detect non-functioning sub-cellular organelles and proteins, they start the so-called nucleation process. The organelles will be surrounded by a double membrane vesicle mainly constituted by endoplasmic reticulum (ER) membrane and autophagy proteins, e.g., MAP1LC3B, Beclin-1, VPS34, Unc-51 like autophagy activating kinase (ULK1) and ubiquitination-related proteins. Then the autophagic membrane will go through an elongation phase involving additional autophagy players. Once the autophagic vesicle is complete, the sub-cellular organelles will be isolated from the rest of the cytosol and driven to the final fusion with lysosomes. Here, the digestion process will end. Alteration and or impairment of autophagy have been shown to be correlated with development of diseases affecting the central nervous system, e.g., Alzheimer and other neurodegenerative diseases. Nonetheless, autophagy defect is responsible for tumorigenesis in blood and solid malignancies, in particular liver cancer. Malignancies of the liver are determined by several genetics and epigenetics mechanisms triggering the up-regulation of survival mechanisms and resistance to cell death. Furthermore, liver cancer could result from pathologic conditions like cirrhosis and fibrosis related to virus infection, aflatoxin, alcohol consumption and high fat diet together with insulin resistance. The role exerted by autophagy in the pathogenesis of the liver and tumor development has been evidenced in recent years. The alteration of autophagy assumes a fundamental role for liver tumorigenesis determining an accumulation of non-functional proteins and organelles that trigger oxidative stress leading to genotoxic stress and gene alterations. Furthermore, the absence of this degradation mechanism could prompt the cells to alter their metabolic status and turn into malignant cells. Interestingly, the heterozygous loss of function of Beclin-1 is able to trigger liver tumorigenesis or even the simple accumulation of proteins caused by the block of the final autolysosome fusion and degradation process is responsible for liver cancer development. This review highlights the importance of targeting the autophagy process in liver cancer in order to restore its function and to promote autophagy-mediated cell demise.

Targeting EGFR-mediated autophagy as a potential strategy for cancer therapy.

Autophagy is a naturally occurring programed cellular catabolic process stimulated by cellular stress for energy homeostasis maintenance and elimination of harmful substances. It mostly works as pro-survival mechanism but on the other hand deregulation of autophagy has been linked to non-apoptotic cell death known as "type II programed cell death." Emerging evidences indicate that EGFR (epidermal growth factor receptor)-mediated RAS/RAF/MEK/ERK signaling pathway plays a critical role in the induction of autophagy in various tumors. It has further been established that this signaling pathway is also involved in several other anti-proliferative events such as apoptosis and senescence. However, the signaling pathway activity and effects are highly dependent on the cell type and the stimulus. It is currently being evident that autophagy induction by RAS/RAF/MEK/ERK pathway through small molecules may be a potential therapeutic strategy for cancer. However, to our best knowledge, the role of EGFR-mediated RAS/RAF/MEK/ERK signaling pathway in autophagy-mediated cell death and survival have not previously been reviewed. In this review, we discuss the current state of knowledge on how RAS/RAF/MEK/ERK signaling pathway regulates autophagy and the role of this EGFR-mediated autophagy in diseases. We further examine the cross-talk between this EGFR-mediated autophagy and apoptosis as well as how this process is currently being utilized for cancer treatment and suggest promoting autophagy-related cell death by small molecules may be exploited to design better therapeutic strategies for early stage and locally advanced tumors.

Invariant BECN1 C‐x‐x‐C Motifs Are Essential For Starvation‐Induced Autophagy

Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1, a protein essential for autophagosome nucleation, contains two invariant C‐x‐x‐C motifs (18CxxC21 and 137CxxC140 in human BECN1) that bookend a large BECN1 intrinsically disordered region (IDR). We use circular dichroism spectroscopy to elucidate differences in secondary structural content of the wild‐type and CysTetrad mutant BECN1 IDR. Additionally, we are using small angle X‐ray scattering analyses to further understand the impact of the CysTetrad mutation on the structure of the BECN1 IDR. We also show that helicity of the BECN1 IDR increases upon addition of 2,2,2‐trifluoroethanol (TFE), suggesting that it may undergo binding‐associated helical transitions. Further, we use ICP‐MS to demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio under both, reducing and non‐reducing environments. Lastly, we show that both C‐x‐x‐C motifs are required, while the IDR is also important, for starvation‐triggered up‐regulation of autophagy.Support or Funding InformationNIH grants RO3 NS090939 and R15 GM122035 (S.C.S.)National Science Foundation grant MCB‐1413525 (S.C.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular response to persistent foot-and-mouth disease virus infection is linked to specific types of alterations in the host cell transcriptome

Food-and-mouth disease virus (FMDV) is a highly contagious virus that seriously threatens the development of animal husbandry. Although persistent FMDV infection can dramatically worsen the situation, the mechanisms involved in persistent FMDV infection remain unclear. In the present study, we identified the presence of evolved cells in the persistently FMDV-infected cell line. These cells exhibited resistance to the parent FMDV and re-established persistent infection when infected with FMDV-Op (virus supernatant of persistent infection cell lines), emphasizing the decisive role of evolved host cells in the establishment of persistent FMDV infection. Using RNA-seq, we identified the gene expression profiles of these evolved host cells. In total, 4,686 genes were differentially expressed in evolved cells compared with normal cells, with these genes being involved in metabolic processes, cell cycle, and cellular protein catabolic processes. In addition, 1,229 alternative splicing events, especially skipped exon events, were induced in evolved cells. Moreover, evolved cells exhibited a stronger immune defensive response and weaker MAPK signal response than normal cells. This comprehensive transcriptome analysis of evolved host cells lays the foundation for further investigations of the molecular mechanisms of persistent FMDV infection and screening for genes resistant to FMDV infection.

De novo transcriptome of the pallial gland of the date mussel (Lithophaga lithophaga)

Lithophaga lithophaga is a rock-boring bivalve with an ability to dissolve carbonate substrata with its siphonal and pallial gland secretions. However, the molecular mechanism that enables this species to bore into calcareous rocks is not yet known. In order to identify genes potentially involved in chemical boring we performed transcriptome sequencing of pallial-gland tissue samples of L. lithophaga. Transcriptome sequencing using an Ion Torrent platform generated 60.563 million clean reads with an average read length of 96 bp. De novo assembly of clean reads produced 62,490 contigs with a mean length of 408 bp. Since the boring mechanism is attributed to calcium-binding proteins, the search focused on transcripts capable of binding this element. In all, 178 genes with calcium-binding ability were found to be expressed in the pallial gland of L. lithophaga. Annexins, calreticulin, phospholipase A2 and V-type proton-ATPases were considered as possible candidate chemical-boring genes due to their known function and involvement in various other biological processes: e.g. ion transport, cellular catabolic process, protein folding. Transcriptome analysis of L. lithophaga revealed a set of candidate genes putatively associated with chemical boring. The selected set of genes will be studied further to verify their expression patterns and their possible involvement in the rate of chemical boring in L. lithophaga.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN’s protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7’s antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Transcriptome analysis reveals differential gene expression in intramuscular adipose tissues of Jinhua and Landrace pigs.

Meat is a rich source of protein, fatty acids and carbohydrates for human needs. In addition to necessary nutrients, high fat contents in pork increase the tenderness and juiciness of the meat, featuring diverse application in various dishes. This study investigated the transcriptomic profiles of intramuscular adipose tissues in Jinhua and Landrace pigs by employing advanced RNA sequencing. Results showed significant interesting to note that there were significant differences in the expression of genes. 1,632 genes showed significant differential expression, 837 genes were up-regulated and 195 genes were down-regulated. Variations in genes responsible for cell aggregation, extracellular matrix formation, cellular lipid catabolic process, and fatty acid binding strongly supported that both pig breeds feature variable fat and muscle metabolism. Certain differentially expressed genes are included in the pathway of mitogen-activated protein kinase signaling pathway, Ras signaling pathway and insulin pathway. Results from real-time quantitative polymerase chain reaction also validated the differential expression of 17 mRNAs between meats of the two pig breeds. Overall, these findings reveal significant differences in fat and protein metabolism of intramuscular adipose tissues of two pig breeds at the transcriptomic level and suggest diversification at the genetic level between breeds of the same species.

The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis

It has been reported that circular RNA (circRNA) is associated with human cancer. However, few studies have been reported in active pulmonary tuberculosis (APTB). The global circRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of APTB patients (n=5) and health controls (HC) (n=5) by using high-throughput sequencing. According to the systematical bioinformatics analysis, the basic content of circRNAs and their fold changes in the two groups were calculated. We selected 6 significant differentially expressed circRNAs, hsa_circ_0005836, hsa_circ_0009128, hsa_circ_0003519, hsa_circ_0023956, hsa_circ_0078768, and hsa_circ_0088452 and validated the expression in PBMCs from APTB (n=10) and HC (n=10) by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Further, the verification of these specific circRNAs (hsa_circ_0005836 and hsa_circ_0009128) between APTB (n=34) and HC (n=30) in PBMCs was also conducted by qRT-PCRs. The RNA-seq data showed the significant differential expression of the 523 circRNAs between the APTB and HC groups (199 circRNAs were significantly up-regulated and 324 circRNAs were down-regulated). Hsa_circ_0005836 and hsa_circ_0009128 expression was significantly down-regulated in the PBMCs of APTB (P<0.05) in the samples of APTB compared to HC in our study. The gene ontology based enrichment analysis of the circRNA-miRNA-mRNAs network showed that cellular catabolic process (P=7.10E-08), regulation of metabolic process (P=2.10E-06), catalytic activity (P=3.67E-08), protein binding (P=1.71E-07), cell part (P=3.46E-06), intracellular part (P=1.71E-07), and intracellular (P=3.67E-08) were recognized in the comparisons between APTB and HC. Based on KEGG analysis, HTLV-I infection, regulation of actin cytoskeleton, neurotrophin signaling pathway and mTOR signaling pathway were relevant during tuberculosis bacillus infection. We found for the first time that hsa_circ_0005836 and hsa_circ_0009128 were significantly down-regulated in the PBMCs of APTB compared with HC. Our findings indicate hsa_circ_0005836 might serve as a novel potential biomarker for TB infection.

Backbone and side chain resonance assignments for a structured domain within Atg32.

Autophagy is a catabolic cellular process that targets cytosolic material, including mitochondria, to the vacuole or lysosomes for degradation. The selective degradation of mitochondria by autophagy is termed mitophagy. Dysfunctional mitophagy, which leads to the accumulation of damaged mitochondria, has been implicated in Parkinson's disease, cancer, cardiac disease and metabolic disease. In Saccharomyces cerevisiae, mitophagy is initiated by the autophagy receptor Atg32, an outer mitochondrial membrane protein. A lack of structural information for Atg32 has hindered our understanding of the molecular mechanisms of mitophagy initiation. To gain new structural insight into Atg32, we have identified the location of a structured domain within the cytosolic region of Atg32 and completed the backbone and side chain resonance assignments for this domain.

The transcriptional response of the Pacific oyster Crassostrea gigas against acute heat stress

The Pacific oyster, Crassostrea gigas, has evolved sophisticated mechanisms to adapt the changing ambient conditions, and protect themselves from stress-induced injuries. In the present study, the expression profiles of mRNA transcripts in the haemocytes of oysters under heat stress were examined to reveal the possible mechanism of heat stress response. There were 23,315, 23,904, 23,123 and 23,672 transcripts identified in the haemocytes of oysters cultured at 25 °C for 0, 6, 12, and 24 h (designed as B, H6, H12, H24), respectively. And 22,330 differentially expressed transcripts (DTs) were yielded in the pairwise comparisons between the above four samples, which corresponded to 8074 genes. There were 9, 12 and 22 Gene Ontology (GO) terms identified in the DT pairwise comparison groups of H6_B, H12_H6 and H24_H12, respectively, and the richest GO terms in biological process category were cellular catabolic process, translational initiation and apoptotic process, respectively. There were 108, 102 and 102 KEGG pathways successfully retrieved from DTs comparison groups DTH6_B, DTH12_H6 and DTH24_H12, respectively, among which 93 pathways were shared by all three comparison groups, and most of them were related to metabolism of protein, carbohydrate and fat. The expression patterns of 12 representative heat stress response-relevant genes detected by quantitative real-time PCR (qRT-PCR) were similar to those obtained from transcriptome analysis. By flow cytometric analysis, the apoptosis rate of haemocytes increased significantly after oysters were treated at 25 °C for 24 h and recovered at 4 °C for 12 h (p < 0.05) and 36 h (p < 0.01), and it also increased significantly when the heat treatment lasted to 60 h (p < 0.01). The present results indicated that, when oysters encountered short term heat stress, the expression of genes related to energy metabolism, as well as unfolded protein response (UPR) and anti-apoptotic system, were firstly regulated to maintain basic life activities, and then a large number of genes involved in stabilizing protein conformation and facilitating further protein refolding were activated to repair the stress injury. However, the stress injury gradually became irreparable with the stress persisting, and apoptosis was activated when the heat treatment prolonged to 24 h. The information was useful to better understand the molecular mechanism of heat stress response and develop strategies for the improvement of oyster survival rate during summer high-temperature period.