Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the 'nucleotide binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7 protein' (NLRP7) inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.

Carbohydrate can be converted into fat by de novo lipogenesis, a process upregulated in fatty liver disease. Chemically, de novo lipogenesis involves polymerization and reduction of acetyl-CoA, using NADPH as the electron donor. The feedstocks used to generate acetyl-CoA and NADPH in lipogenic tissues remain, however, unclear. Here we show using stable isotope tracing in mice that de novo lipogenesis in adipose is supported by glucose and its catabolism via the pentose phosphate pathway to make NADPH. The liver, in contrast, derives acetyl-CoA for lipogenesis from acetate and lactate, and NADPH from folate-mediated serine catabolism. Such NADPH generation involves the cytosolic serine pathway in liver running in the opposite direction to that observed in most tissues and tumours, with NADPH made by the SHMT1-MTHFD1-ALDH1L1 reaction sequence. SHMT inhibition decreases hepatic lipogenesis. Thus, liver folate metabolism is distinctively wired to support cytosolic NADPH production and lipogenesis. More generally, while the same enzymes are involved in fat synthesis in liver and adipose, different substrates are used, opening the door to tissue-specific pharmacological interventions.

Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive condition characterized by the impairment of alveolar epithelial cells. Despite continued research efforts, the effective therapeutic medication is still absent due to an incomplete understanding of the underlying etiology. It has been shown that rhythmic alterations are of significant importance in the pathophysiology of IPF. However, a comprehensive understanding of how metabolite level changes with circadian rhythms in individuals with IPF is lacking. Here, we constructed an extensive metabolite database by utilizing an unbiased reference system culturing with 13C or 15N labeled nutrients. Using LC-MS analysis via ESI and APCI ion sources, 1300 potential water-soluble metabolites were characterized and applied to evaluate the metabolic changes with rhythm in the lung from both wild-type mice and mice with IPF. The metabolites, such as glycerophospholipids and amino acids, in WT mice exhibited notable rhythmic oscillations. The concentrations of phospholipids reached the highest during the fast state, while those of amino acids reached their peak during fed state. Similar diurnal variations in the metabolite rhythm of amino acids and phospholipids were also observed in IPF mice. Although the rhythmic oscillation of metabolites in the urea cycle remained unchanged, there was a significant up-regulation in their levels in the lungs of IPF mice. 15N-ammonia in vivo isotope tracing further showed an increase in urea cycle activity in the lungs of mice with IPF, which may compensate for the reduced efficiency of the hepatic urea cycle. In sum, our metabolomics database and method provide evidence of the periodic changes in lung metabolites, thereby offering valuable insights to advance our understanding of metabolic reprogramming in the context of IPF.

Mycobacterium avium subspecies paratuberculosis (Map) causes Johne's disease in domestic and wild ruminants. It has been a debate that whether Map can cause Crohn's disease in human. To our knowledge there is no report about molecular characterization of Map in China, although several Map strains have been reported in other country. The objectives of this study was to know the recent prevalence of Johne's disease in dairy farms in Shandong province, and have a better understanding of genotypic distribution of Map in China.

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine-addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low-invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high-invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients' microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high-invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low-invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC.

Myocardial infarction is a clinical form of necrosis in the myocardium caused by an imbalance between the coronary blood supply and myocardial demand. Garcinol is a polyisoprenylated benzophenone found in the fruit of Garcinia indica, which is abundant in tropical regions. This fruit contains high levels of garcinol, isoxanthochymol, isogarcinol, hydroxycitric acid and xanthochymol. Garcinol and hydroxycitric acid have been shown to have antioxidant effects. In this study, rats were assigned to sham, control, low-dose, high-dose and positive control groups. Hemodynamic and apoptotic markers were evaluated, and histopathological analysis was conducted. The mRNA and protein levels of caspase-3, Bax, Bcl-2 and cleaved caspase-3 were quantified. Garcinol treatment increased the heart rate and improved the maximum rate of increase in left-ventricle (LV) pressure (+dp/dtmax), maximum rate of decrease in LV pressure (-dp/dtmax), LV ejection fraction and LV systolic pressure in rats with induced heart failure. Garcinol treatment reversed body, liver and heart weight changes, resulting in returns to near-normal levels. In the garcinol treatment group, the number of broken fibers, extent of inflammatory cell infiltration and rate of apoptosis remained within normal ranges. Garcinol reduced the cross-sectional areas of cardiomyocytes, and reduced interstitial fibrosis to a normal level. The mRNA and protein levels of cleaved caspase-3, caspase-3 and Bax were reduced, whereas those of Bcl-2 were increased, following high-dose (100 mg/kg) garcinol treatment. These findings suggest that garcinol effectively prevents apoptosis in rats with isoproterenol-induced heart failure and in cardiac H9C2 cells.

NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

Prion diseases are a group of infectious neurodegenerative diseases characterized by multiple neuropathological hallmarks, including accumulation of PrPSc, synaptic damage, and neuronal death. We previously reported that the repressor element 1-silencing transcription factor (REST), a novel neuroprotective marker in neurodegeneration, protects neurons against neurotoxic peptide (PrP106-126)-induced neurotoxicity, but fails to maintain survival following prolonged exposure to PrP106-126. Because Wnt signaling partially induces REST and is activated by lithium, we investigated the effects of lithium on REST in prion diseases. Lithium restores nuclear expression of REST, which is essential for regulating survival proteins. Lithium also mimics neuroprotective functions when REST is blocked, and these beneficial effects are additive with REST overexpression under physiological conditions. Reciprocally, under PrP106-126-stimulated pathological conditions, REST plays a critical role in the neuroprotective mechanisms of lithium treatment. Although lithium recovers Wnt signaling by inhibiting glycogen synthase kinase-3β and stabilizing β-catenin, restores survival associated proteins after exposure to PrP106-126 in primary cortical neurons. Knockdown of REST significantly suppresses the neuroprotective function of lithium. Conversely, overexpression of REST partially recovers its actions. Notably, lithium directly alleviates PrP106-126-induced synaptic damage and neuronal cell death by preventing changes in presynaptic and postsynaptic marker proteins and promoting survival pathways also partially via the expression of REST. Our results suggest that REST acts as a novel and important nuclear target for lithium. We hypothesize that PrP106-126-stimulated neurotoxicity induces Wnt signaling dysfunction and lithium mimics this signaling cascade, suggesting that lithium should be considered as a potential therapeutic agent against prion diseases.

Protein-protein interactions (PPIs) play a crucial role in various biological processes. To better comprehend the pathogenesis and treatments of various diseases, it is necessary to learn the detail of these interactions. However, the current experimental method still has many false-positive and false-negative problems. Computational prediction of protein-protein interaction has become a more important prediction method which can overcome the obstacles of the experimental method. In this work, we proposed a novel computational domain-based method for PPI prediction, and an SVM model for the prediction was built based on the physicochemical property of the domain. The outcomes of SVM and the domain-domain score were used to construct the prediction model for protein-protein interaction. The predicted results demonstrated the domain-based research can enhance the ability to predict protein interactions.

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C) in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C) in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C) promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C) suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C) as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

Dysregulated energetic metabolism has been recently identified as a hallmark of cancer. Although mutations in metabolic enzymes hardwire metabolism to tumourigenesis, they are relatively infrequent in ovarian cancer. More often, cancer metabolism is re-engineered by altered abundance and activity of the metabolic enzymes. Here we identify ubiquitin-specific peptidase 13 (USP13) as a master regulator that drives ovarian cancer metabolism. USP13 specifically deubiquitinates and thus upregulates ATP citrate lyase and oxoglutarate dehydrogenase, two key enzymes that determine mitochondrial respiration, glutaminolysis and fatty acid synthesis. The USP13 gene is co-amplified with PIK3CA in 29.3% of high-grade serous ovarian cancers and its overexpression is significantly associated with poor clinical outcome. Inhibiting USP13 remarkably suppresses ovarian tumour progression and sensitizes tumour cells to the treatment of PI3K/AKT inhibitor. Our results reveal an important metabolism-centric role of USP13, which may lead to potential therapeutics targeting USP13 in ovarian cancers.

Mycobacteria can trigger the AIM2 inflammasome, autophagy activation and type-I interferon release, which are both activated by cytosolic DNA. We have recently demonstrated that activation of the AIM2 inflammasome during M. bovis infection is the result of mycobacterial translocation into the cytosol. To elucidate the effects of inflammasome activation on autophagy, we investigated the role of the AIM2 inflammasome from macrophages infected with a virulent strain of M. bovis. The results showed that the M. bovis-induced AIM2 inflammasome activation decreases autophagy in immortalized and primary murine macrophages. This relied on the inflammasome sensor AIM2 which conjugates with cytosolic DNA to inhibit the STING-dependent pathway involved in selective autophagy and interferon-β release in Mycobacterium-infected macrophages. These results suggest that the AIM2 cytosolic DNA sensor may conjugate competitively with cytosolic M. bovis DNA to restrict M. bovis induced STING-TBK1-dependent autophagy activation and IFN-β secretion.

M-ficolin, a pathogen recognition molecule in the innate immune system, binds sugar residues including N-acetyl-D-glucosamine (GlcNAc), which is displayed on invading microbes and on apoptotic cells. The cis and trans Asp282-Cys283 peptide bond in the M-ficolin, which was found to occur at neutral and acidic pH in crystal structures, has been suggested to represent binding and non-binding activity, respectively. A detailed understanding of the pH-dependent conformational changes in M-ficolin and pH-mediated discrimination mechanism of GlcNAc-binding activity are crucial to both immune-surveillance and clearance of apoptotic cells.

Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL)-1β release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1β in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in IL-1β release from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126).

Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles; this process also eliminates pathological PrPSc as it accumulates during prion infection. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated. In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene-silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signaling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation. Our findings suggest that autophagy up-regulation and inhibition of Caspase-1 may protect against prion-induced neuroinflammation and accelerate misfolded protein degradation and are potential therapeutic approaches for prion diseases.