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1.  Linking adiponectin and autophagy in the regulation of breast cancer metastasis 
Adipokines within the tumor microenvironment may play important roles in regulating the early steps of breast cancer metastasis. Adiponectin (AdipoQ) is the most abundant adipokine and exists in multiple forms: full-length multimers (fAd) and a cleaved, globular isoform (gAd). While these isoforms are observed as having distinct biological properties, nearly all investigation into AdipoQ in breast cancer has focused on the anti-tumor roles of fAd, while mostly ignoring gAd. However, evidence from other disease settings suggests that gAd is linked to processes known to promote metastasis. Here we discuss key areas in which knowledge about AdipoQ in breast cancer is lacking, expressly focusing on data suggesting that gAd is elevated in the microenvironment and may act directly on invasive breast cancer cells to support their initial metastatic progression. We discuss autophagy as a potential mechanism of action for this effect. Overall, given that AdipoQ and AdipoQ receptor agonists have been proposed as therapeutic strategies, it is necessary to better understand the various functions of these regulatory molecules in metastatic breast cancer. Doing so will help ensure the most effective approaches to treating this disease, for which there remain no curative options.
PMCID: PMC4197061  PMID: 24903246
Adiponectin; autophagy; breast cancer; metastasis
2.  Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1 
Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD.
PMCID: PMC4197071  PMID: 24984783
Autosomal recessive polycystic kidney disease; PKHD1; Alternative splicing; Exon splice enhancers
3.  Antagonism of angiotensin 1–7 prevents the therapeutic effects of recombinant human ACE2 
Activation of the angiotensin 1–7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1–7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1–7 action. Wild type male C57BL/6 mice (10–12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1–7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1–7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1–7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1–7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1–7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1–7, and increased Ang 1–7 action represents a potential therapeutic strategy for cardiovascular diseases.
PMCID: PMC4580513  PMID: 25874965
Renin–angiotensin system; Angiotensin-converting enzyme 2; Angiotensin 1–7; PI3K/Akt signaling
4.  Role of Macrophage Sialoadhesin in Host Defense Against the Sialylated Pathogen Group B Streptococcus 
Several bacterial pathogens decorate their surfaces with sialic acid residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic-acid binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, sialoadhesin is a unique Siglec with an elongated structure that lacks intracellular signaling motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via sialoadhesin-sialic acid mediated recognition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restricted the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in sialoadhesin-deficient mice. Thus sialoadhesin represents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens.
PMCID: PMC4133643  PMID: 24788876
metallophillic macrophage; antibody; siglec; sialic acid
5.  Activation of the EGFR/p38/JNK Pathway by Mitochondrial-Derived Hydrogen Peroxide Contributes To Oxygen-induced Contraction Of Ductus Arteriosus 
Oxygen-induced contraction of the ductus arteriosus (DA) involves a mitochondrial oxygen-sensor, which signals pO2 in the DA smooth muscle cell (DASMC) by increasing production of diffusible hydrogen peroxide (H2O2). H2O2 stimulates vasoconstriction by regulating ion channels and rho kinase, leading to calcium influx and calcium sensitization. Because epidermal growth factor receptor (EGFR) signaling is also redox regulated and participates in oxygen sensing and vasoconstriction in other systems, we explored the role of the EGFR and its signaling cascade (p38 and JNK) in DA contraction.
Experiments were performed in DA rings isolated from full-term New Zealand White rabbits and human DASMC. In human DASMCs increasing pO2 from hypoxia to normoxia (40 to 100 mmHg) significantly increased cytosolic calcium, p<0.01. This normoxic rise in intracellular calcium was mimicked by EGF and inhibited by EGFR siRNA. In DA rings, EGF caused contraction whilst the specific EGFR inhibitor (AG1478) and the tyrosine kinase inhibitors (genistein or tyrphostin A23) selectively attenuated oxygen-induced contraction (p <0.01). Conversely, orthovanadate, a tyrosine phosphatase inhibitor known to activate EGFR signaling, caused dose-dependent contraction of hypoxic DA and superimposed increases in oxygen caused minimal additional contraction. Ansomycin, an activator of EGFR’s downstream kinases, p38 and JNK, caused DA contraction; conversely, oxygen-induced DA contraction was blocked by inhibitors of p38 MAPK (SB203580) or JNK (JNK inhibitor II). O2-induced phosphorylation of EGFR occurred within 5-minutes of increasing pO2 and was inhibited by mitochondrial-targeted overexpression of catalase. AG1478 prevented the oxygen-induced p38 and JNK phosphorylation. In conclusion, O2-induced EGFR transactivation initiates p38/JNK-mediated increases in cytosolic calcium and contributes to DA contraction. The EGFR/p38/JNK pathway is regulated by mitochondrial redox signaling and is a promising therapeutic target for modulation of the patent ductus arteriosus.
PMCID: PMC4134409  PMID: 24906456
Oxygen sensing; tyrosine kinase inhibitors; c-Jun N-amino-terminal kinase (JNK); Patent ductus arteriosus (PDA); protein tyrosine phosphatases; Mitochondrial hydrogen peroxide
6.  The Role of HIF1α in Renal Cell Carcinoma Tumorigenesis 
The transcription factor HIF1α is implicated in the development of clear cell renal cell carcinoma (ccRCC). Although HIF1α was initially believed to be essential for ccRCC development, recent studies hypothesize an oncogenic role for HIF2α in ccRCC, but a tumor suppressor role for HIF1α [1], leading to uncertainty as to the precise roles of the different HIF transcription factors in this disease.
Using evidence available from studies with human ccRCC cell lines, mouse xenografts, murine models of ccRCC, and human ccRCC specimens, we evaluate the roles of HIF1α and HIF2α in the pathogenesis of ccRCC. We present a convergence of clinical and mechanistic data supporting an important role for HIF1α in promoting tumorigenesis in a clinically important and large subset of ccRCC. This indicates that current understanding of the exact roles of HIF1α and HIF2α is incomplete and that further research is required to determine the diverse roles of HIF1α and HIF2α in ccRCC.
PMCID: PMC4119538  PMID: 24916472
Clear cell renal cell carcinoma; Cancer stem cells; Hypoxia inducible factor 1α; Kidney cancer; Carcinogenesis; Review
7.  Chemokines and their receptors in Atherosclerosis 
Atherosclerosis, a chronic inflammatory disease of the medium- and large-sized arteries, is the main underlying cause of cardiovascular diseases (CVDs) most often leading to a myocardial infarction or stroke. However, atherosclerosis can also develop without this clinical manifestation. The pathophysiology of atherosclerosis is very complex and consists of many cells and molecules interacting with each other. Over the last years, chemokines (small 8–12 kDa cytokines with chemotactic properties) have been identified as key players in atherogenesis. However, this remains a very active and dynamic field of research. Here, we will give an overview of the current knowledge about the involvement of chemokines in all phases of atherosclerotic lesion development. Furthermore, we will focus on two chemokines that recently have been associated with atherogenesis, CXCL12, and macrophage migration inhibitory factor (MIF). Both chemokines play a crucial role in leukocyte recruitment and arrest, a critical step in atherosclerosis development. MIF has shown to be a more pro-inflammatory and thus pro-atherogenic chemokine, instead CXCL12 seems to have a more protective function. However, results about this protective role are still quite debatable. Future research will further elucidate the precise role of these chemokines in atherosclerosis and determine the potential of chemokine-based therapies.
PMCID: PMC4577534  PMID: 26175090
Cardiovascular disease; Atherosclerosis; Chemokines; Macrophage migration inhibitory factor; CXCL12
8.  Molecules in medicine mini review: the αβ T cell receptor 
As an integral part of the mammalian immune system, a distributed network of tissues, cells, and extra-cellular factors, T lymphocytes perform and control a multitude of activities that collectively contribute to the effective establishment, maintenance, and restoration of tissue and organismal integrity. Development and function of T cells is controlled by the T cell receptor (TCR), a heterodimeric cell surface protein uniquely expressed on T cells. During T cell development, the TCR undergoes extensive somatic diversification that generates a diverse T cell repertoire capable of recognizing an extraordinary range of protein and nonprotein antigens presented in the context of major histocompatibility complex molecules (MHC). In this review, we provide an introduction to the TCR, describing underlying principles that position this molecule as a central regulator of the adaptive immune system involved in responses ranging from tissue protection and preservation to pathology and autoimmunity.
PMCID: PMC4269364  PMID: 24848996
Tcell receptor; Major histocompatibility complex; Tcell repertoire
9.  The pneumococcus: why a commensal misbehaves 
Several characteristics of Streptococcus pneumoniae (pneumococcus) combine to make it a particularly problematic pathogen. Firstly, the pneumococcus has the capacity to cause disease through the expression of virulence factors such as its polysaccharide capsule and pore-forming toxin. In addition, the pneumococcus is highly adaptable as demonstrated by its ability to acquire and disseminate resistance to multiple antibiotics. Although the pneumococcus is a major cause of disease, the organism is most commonly an “asymptomatic” colonizer of its human host (the carrier state), with transmission occurring exclusively from this reservoir of commensal organisms. Thus, it is unclear how the organism’s virulence and adaptability promote its persistence or host to host spread during its carrier state. This review summarizes current understanding of how these characteristics may contribute to the commensal lifestyle of the pneumococcus.
PMCID: PMC4487619  PMID: 19898768
Colonization; Virulence factor; Neutrophil; Pneumococcus
10.  Ubiquitous Points of Control over Regulatory T cells 
Posttranslational modification by ubiquitin tagging is crucial for regulating the stability, activity and cellular localization of many target proteins and processes including DNA repair, cell cycle progression, protein quality control and signal transduction. It has long been appreciated that ubiquitin-mediated events are important for certain signaling pathways leading to leukocyte activation and the stimulation of effector function. It is now clear that the activities of molecules and pathways central to immune regulation are also modified and regulated through ubiquitin. Among the mechanisms of immune control, regulatory T cells (or Tregs) are themselves particularly sensitive to such regulation. E3 ligases and deubiquitinases both influence Tregs through their effects on signaling pathways pertinent for these cells or through the direct, posttranslational regulation of Foxp3. In this review we will summarize and discuss several examples of ubiquitin-mediated control over multiple aspects of biology of Tregs including their generation, function and phenotypic fidelity. Fully explored and exploited, these potential opportunities for Treg modulation may lead to novel immunotherapies for both positive and negative fine-tuning of immune restraint.
PMCID: PMC4083097  PMID: 24777637
E3 ligase; Foxp3; Treg; Ubiquitin; Deubiquitinase
11.  Control of Autophagy Maturation by Acid Sphingomyelinase in Mouse Coronary Arterial Smooth Muscle Cells: Protective Role in Atherosclerosis 
Recent studies have indicated a protective role of autophagy in regulating vascular smooth muscle cells homeostasis in atherogenesis, but the mechanisms controlling autophagy, particularly autophagy maturation, are poorly understood. Here, we investigated whether acid sphingomyelinase (ASM)-regulated lysosome function is involved in autophagy maturation in coronary arterial smooth muscle cells (CASMCs) in the pathogenesis of atherosclerosis. In coronary arterial wall of ASM-deficient (Smpd1−/−) mice on Western diet, there were high expression levels of both LC3B, a robust marker of autophagosomes (APs), and p62, a selective autophagy substrate, compared to those in wild type (Smpd1+/+) mice. By Western blotting and flow cytometry, atherogenic stimulation of Smpd1+/+ CASMCs with 7-ketocholesterol was found to significantly enhance LC3B expression and increase the content of both APs and autophagolysosomes (APLs). In Smpd1−/− CASMCs, such 7-ketocholesterol-induced increases in LC3B and p62 expression and APs were further augmented, but APLs formation was abolished. Analysis of fluorescence resonance energy transfer (FRET) between fluorescence-labeled LC3B and Lamp1 (lysosome marker) showed that 7-ketocholesterol markedly induced fusion of APs with lysosomes in Smpd1+/+ CASMCs, which was abolished in Smpd1−/− CASMCs. Moreover, 7-ketocholesterol-induced expression of cell dedifferentiation marker vimentin and proliferation was enhanced in Smpd1−/− CASMCs compared to those in Smpd1+/+ CASMCs. Lastly, overexpression of ASM further increased APLs formation in Smpd1+/+ CASMCs and restored APLs formation in Smpd1−/− CASMCs indicating that increased ASM expression is highly correlated with enhanced APLs formation. Taken together, our data suggest that the control of lysosome trafficking and fusion by ASM is essential to a normal autophagic flux in CASMCs, which implicates that the deficiency of ASM-mediated regulation of autophagy maturation may result in imbalance of arterial smooth muscle cell homeostasis and thus serve as an important atherogenic mechanism in coronary arteries.
PMCID: PMC4211081  PMID: 24463558
Acid sphingomyelinase; autophagic flux; lysosome; coronary arterial smooth muscle cell; coronary artery; atherosclerosis
Evolution has long provided a foundation for population genetics, but many major advances in evolutionary biology from the 20th century are only now being applied in molecular medicine. They include the distinction between proximate and evolutionary explanations, kin selection, evolutionary models for cooperation, and new strategies for tracing phylogenies and identifying signals of selection. Recent advances in genomics are further transforming evolutionary biology and creating yet more opportunities for progress at the interface of evolution with genetics, medicine, and public health. This article reviews 15 evolutionary principles and their applications in molecular medicine in hopes that readers will use them and others to speed the development of evolutionary molecular medicine.
PMCID: PMC4416654  PMID: 22544168
Evolution; biology; genetics; Darwinian medicine; evolutionary medicine
13.  miR-26a enhances autophagy to protect against ethanol-induced acute liver injury 
Autophagy is a process for the turnover of intracellular organelles and molecules during stress responses. microRNAs (miRNAs) are small, non-coding endogenous RNAs that may regulate almost every cellular process. However, the roles of miRNAs in autophagy are still poorly understood. In this study, we show that miR-26a enhances autophagy in both culture cells and the mouse liver. Hepatic overexpression of miR-26a in mice alleviated ethanol-induced hepatic steatosis and liver injury. Overexpression of miR-26a increased the expression of the autophagy mediator Beclin-1, which is regulated by mitogen-activated protein kinases (MAPKs). We identified DUSP4 and DUSP5, two MAPKs inhibitors, as direct targets of miR-26a. We further demonstrated that miR-26a targeted the 3′-UTRs of several other negative regulators of autophagy. Our results thus identify a novel miRNA-mediated mechanism that enhances cytoprotective autophagy in the liver.
Key messages
• miR-26a enhances autophagy in liver cells.
• Hepatic overexpression of miR-26a in mice alleviates ethanol-induced liver injury.
• Overexpression of miR-26a increases the expression of autophagy mediator Beclin-1.
• DUSP4 and DUSP5, two MAPKs inhibitors, were identified as direct targets of miR-26a.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-015-1282-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4577542  PMID: 25877859
miR-26a; Autophagy; Hepatic steatosis; Mitogen-activated protein kinases; Ethanol binge
14.  Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance 
Alzheimer’s disease (AD) is a neurodegenerative disorder associated with amyloid accumulation and autophagic changes. Parkin is an E3 ubiquitin ligase involved in proteasomal and autophagic clearance. We previously demonstrated decreased parkin solubility and interaction with the key autophagy enzyme Beclin-1 in AD, but tyrosine kinase inhibition restored parkin-Beclin-1 interaction. In the current studies we determined the mechanisms of Nilotinib-induced parkin-Beclin-1 interaction, which leads to amyloid clearance. Nilotinib increased endogenous parkin levels and ubiquitination, which may enhance parkin recycling via the proteasome, leading to increased activity and interaction with Beclin-1. Parkin solubility was decreased and autophagy was altered in amyloid expressing mice, suggesting that amyloid stress affects parkin stability, leading to failure of protein clearance via the lysosome. Isolation of autophagic vacuoles revealed amyloid and parkin accumulation in autophagic compartments but Nilotinib decreased insoluble parkin levels and facilitated amyloid deposition into lysosomes in wild type, but not parkin−/− mice, further underscoring an essential role for endogenous parkin in amyloid clearance. These results suggest that Nilotinib boosts the autophagic machinery, leading to increased level of endogenous parkin that undergoes ubiquitination and interacts with Beclin-1 to facilitate amyloid clearance. These data suggest that Nilotinib-mediated autophagic changes may trigger parkin response via increased protein levels, providing a therapeutic strategy to reduce Aβ and Tau in AD.
PMCID: PMC3975659  PMID: 24337465
Ubiquitination; parkin; autophagy; Tau; amyloid; Alzheimer’s
15.  Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage 
Photooxidative damage and chronic innate immune activation have been implicated in retinal pigment epithelium (RPE) dysfunction, a process that underlies blinding diseases such as age-related macular degeneration (AMD). To identify a potential molecular link between these mechanisms, we investigated whether lipofuscin-mediated phototoxicity activates the NLRP3 inflammasome in RPE cells in vitro. We found that blue light irradiation (dominant wavelength 448 nm, irradiance 0.8 mW/cm2, duration 6 h) of lipofuscin-loaded primary human RPE cells and ARPE-19 cells induced photooxidative damage, lysosomal membrane permeabilization (79.5 % of cells vs. 3.8 % in nonirradiated controls), and cytosolic leakage of lysosomal enzymes. This resulted in activation of the inflammasome with activation of caspase-1 and secretion of interleukin-1β (14.6 vs. 0.9 pg/ml in nonirradiated controls) and interleukin-18 (87.7 vs. 0.2 pg/ml in nonirradiated controls). Interleukin secretion was dependent on the activity of NLRP3, caspase-1, and lysosomal proteases cathepsin B and L. These results demonstrate that accumulation of lipofuscin-like material in vitro renders RPE cells susceptible to phototoxic destabilization of lysosomes, resulting in NLRP3 inflammasome activation and secretion of inflammatory cytokines. This new mechanism of inflammasome activation links photooxidative damage and innate immune activation in RPE pathology and may provide novel targets for therapeutic intervention in retinal diseases such as AMD.
Key message
• Visible light irradiation of lipofuscin-loaded RPE cells activates inflammasome.
• Inflammasome activation results from lysosomal permeabilization and enzyme leakage.
• Inflammasome activation induces secretion of inflammatory cytokines by RPE cells.
• Photooxidative damage by visible light as new mechanism of inflammasome activation.
• Novel link between hallmark pathogenetic features of retinal degenerative diseases.
PMCID: PMC4510924  PMID: 25783493
Age-related macular degeneration; Retinal pigment epithelium; Interleukin-1β; Lysosomal membrane permeabilization; Lipid peroxidation
16.  A novel COCH mutation associated with autosomal dominant nonsyndromic hearing loss disrupts the structural stability of the vWFA2 domain 
Mutations in COCH have been associated with autosomal dominant nonsyndromic hearing loss (DFNA9) and are frequently accompanied by vestibular hypofunction. Here, we report identification of a novel missense mutation, p.F527C, located in the vWFA2 domain in members of a Korean family with late-onset and progressive hearing loss. To assess the molecular characteristics of this cochlin mutant, we constructed both wild-type and mutant cochlin constructs and transfected these into mammalian cell lines. Results of immunocytochemistry analysis demonstrated localization of the cochlin mutant in the ER/Golgi complex, whereas western blot analyses of cell lysates revealed that the mutant cochlin tends to form covalent complexes that are retained in the cell. Biochemical analyses of recombinant vWFA2 domain of cochlin carrying the p.F527C mutation revealed that the mutation increases propensity of the protein to form covalent disulfide-bonded dimers and affects the structural stability but not the collagen-affinity of the vWFA2 domain. We suggest that the instability of mutant cochlin is the major driving force for cochlin aggregation in the inner ear in DFNA9 patients carrying the COCH p.F527C mutation.
PMCID: PMC4361775  PMID: 22610276
nonsyndromic hearing loss; DFNA9; cochlin; mutation; protein stability
17.  Metal-deficient SOD1 in amyotrophic lateral sclerosis 
Mutations to the ubiquitous antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) were the first established genetic cause of the fatal, adult-onset neurodegenerative disease amyotrophic lateral sclerosis (ALS). It is widely accepted that these mutations do not cause ALS via a loss of antioxidant function, but elucidating the alternate toxic gain of function has proven to be elusive. Under physiological conditions, SOD1 binds one copper ion and one zinc ion per monomer to form a highly stable and functional homodimer, but there is now ample evidence to indicate aberrant persistence of SOD1 in an intermediate metal-deficient state may contribute to the protein’s involvement in ALS. This review briefly discusses some of the data to support a role for metal-deficient SOD1 in the development of ALS and some of the outcomes from drug development studies that have aimed to modify the symptoms of ALS by targeting the metal state of SOD1. The implications for the metal state of SOD1 in cases of sporadic ALS that do not involve mutant SOD1 are also discussed.
PMCID: PMC4408375  PMID: 25754173
Amyotrophic lateral sclerosis (ALS); Motor neuron disease (MND); Copper (Cu); Zinc (Zn); Cu/Zn superoxide dismutase (SOD1); Protein misfolding; Diacetylbis(4-methylthiosemicarbazonato)copperII
18.  NLRP3 inflammasome activation is required for fibrosis development in NAFLD 
NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3−/− mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3−/−mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease.
PMCID: PMC4349416  PMID: 24861026
NLRP3; Inflammation; Liver fibrosis; NASH; Steatoheptatitis
19.  MitoTimer: A Novel Protein for Monitoring Mitochondrial Turnover 
Mitochondrial quality control refers to the coordinated cellular systems involved in maintaining a population of healthy mitochondria. In addition to mitochondrial protein chaperones (Hsp10, Hsp60 and others) and proteases (Lon, AAA proteases) needed for refolding or degrading individual proteins, mitochondrial integrity is maintained through regulation of protein import via the TOM/TIM complex and protein redistribution across the network via fusion and fission; and through mitophagy and biogenesis, key determinants of mitochondrial turnover. A growing number of studies point to the importance of mitochondrial dynamics (fusion/fission) and mitochondrial autophagy in the heart. Mitochondrial biogenesis must keep pace with mitophagy in order to maintain a stable number of mitochondria. In this review we will discuss the use of MitoTimer as a tool to monitor mitochondrial turnover.
PMCID: PMC4333239  PMID: 25479961
20.  Complement evasion by Bordetella pertussis: implications for improving current vaccines 
Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.
PMCID: PMC4366546  PMID: 25686752
Complement; Bordetella pertussis; Innate immunity; Evasion; Vaccine
21.  Both GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells 
Mitochondrial glutaminase (GA) plays an essential role in cancer cell metabolism, contributing to biosynthesis, bioenergetics and redox balance. Humans contain several GA isozymes encoded by the GLS and GLS2 genes, but the specific roles of each in cancer metabolism are still unclear. In this study, glioma SFxL and LN229 cells with silenced isoenzyme glutaminase KGA (encoded by GLS) showed lower survival ratios and a reduced GSH-dependent antioxidant capacity. These GLS-silenced cells also demonstrated induction of apoptosis indicated by enhanced annexin V binding capacity and caspase 3 activity. GLS silencing was associated with decreased mitochondrial membrane potential (ΔΨm) (JC-1 dye test), indicating that apoptosis was mediated by mitochondrial dysfunction. Similar observations were made in T98 glioma cells overexpressing glutaminase isoenzyme GAB, encoded by GLS2, though some characteristics (GSH/GSSG ratio) were different in the differently treated cell lines. Thus, control of GA isoenzyme expression may prove to be a key tool to alter both metabolic and oxidative stress in cancer therapy. Interestingly, reactive oxygen species (ROS) generation by treatment with oxidizing agents: arsenic trioxide or hydrogen peroxide, synergizes with either KGA silencing or GAB overexpression to suppress malignant properties of glioma cells, including the reduction of cellular motility. Of note, negative modulation of GLS isoforms or GAB overexpression evoked lower c-myc and bcl-2 expression, as well as higher pro-apoptotic bid expression. Combination of modulation of GA expression and treatment with oxidizing agents may become a therapeutic strategy for intractable cancers and provides a multi-angle evaluation system for anti-glioma pre-clinical investigations.
PMCID: PMC4327995  PMID: 24276018
Apoptosis; Cancer; Glioma; Glutaminase; Glutathione; ROS
22.  Cancer genomics: why rare is valuable 
Rare conditions are sometimes ignored in biomedical research because of difficulties in obtaining specimens and limited interest from fund raisers. However, the study of rare diseases such as unusual cancers has again and again led to breakthroughs in our understanding of more common diseases. It is therefore unsurprising that with the development and accessibility of next-generation sequencing, much has been learnt from studying cancers that are rare and in particular those with uniform biological and clinical behavior. Herein, we describe how shotgun sequencing of cancers such as granulosa cell tumor, endometrial stromal sarcoma, epithelioid hemangioendothelioma, ameloblastoma, small-cell carcinoma of the ovary, clear-cell carcinoma of the ovary, nonepithelial ovarian tumors, chondroblastoma, and giant cell tumor of the bone has led to rapidly translatable discoveries in diagnostics and tumor taxonomies, as well as providing insights into cancer biology.
PMCID: PMC4366545  PMID: 25676695
Next-generation sequencing; Formefruste; Rare tumors; Genomics
23.  Mitochondrial biogenesis in the metabolic syndrome and cardiovascular disease 
The metabolic syndrome is a constellation of metabolic disorders including obesity, hypertension, and insulin resistance, components which are risk factors for the development of diabetes, hypertension, cardiovascular, and renal disease. Pathophysiological abnormalities that contribute to the development of the metabolic syndrome include impaired mitochondrial oxidative phosphorylation and mitochondrial biogenesis, dampened insulin metabolic signaling, endothelial dysfunction, and associated myocardial functional abnormalities. Recent evidence suggests that impaired myocardial mitochondrial biogenesis, fatty acid metabolism, and antioxidant defense mechanisms lead to diminished cardiac substrate flexibility, decreased cardiac energetic efficiency, and diastolic dysfunction. In addition, enhanced activation of the renin–angiotensin–aldosterone system and associated increases in oxidative stress can lead to mitochondrial apoptosis and degradation, altered bioenergetics, and accumulation of lipids in the heart. In addition to impairments in metabolic signaling and oxidative stress, genetic and environmental factors, aging, and hyperglycemia all contribute to reduced mitochondrial biogenesis and mitochondrial dysfunction. These mitochondrial abnormalities can predispose a metabolic cardiomyopathy characterized by diastolic dysfunction. Mitochondrial dysfunction and resulting lipid accumulation in skeletal muscle, liver, and pancreas also impede insulin metabolic signaling and glucose metabolism, ultimately leading to a further increase in mitochondrial dysfunction. Interventions to improve mitochondrial function have been shown to correct insulin metabolic signaling and other metabolic and cardiovascular abnormalities. This review explores mechanisms of mitochondrial dysfunction with a focus on impaired oxidative phosphorylation and mitochondrial biogenesis in the pathophysiology of metabolic heart disease.
PMCID: PMC4319704  PMID: 20725711
Metabolic impairment; Oxidative phosphorylation; Mitochondrial biogenesis
24.  New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases 
The processes that control the number and shape of the mitochondria (mitochondrial dynamics) and the removal of damaged mitochondria (mitophagy) have been the subject of intense research. Recent work indicates that these processes may contribute to the pathology associated with cardiac diseases. This review describes some of the key proteins that regulate these processes and their potential as therapeutic targets for cardiac diseases.
PMCID: PMC4333238  PMID: 25652199
Cardiac disease; Mitochondria; Mitophagy; Autophagy; Fission; Fusion; Mitochondrial dynamics; Heart
25.  [No title available] 
PMCID: PMC3926703  PMID: 24297496

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