ABSTRACT
Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.
Subject(s)
AMP-Activated Protein Kinases , Carbon Monoxide , Mice , Animals , Phosphorylation , Carbon Monoxide/metabolism , AMP-Activated Protein Kinases/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Fibroblasts/metabolism , Heme/metabolism , Adenosine Triphosphate/metabolismABSTRACT
Intracellular carbon monoxide (CO) is a gaseous signaling molecule and is generated enzymatically by heme oxygenases upon degradation of heme to billiverdin. Target structures for intracellular produced CO are heme proteins including cytochrome c oxidase of the respiratory chain, cytochrome P450-dependent monooxygenases, or myoglobin. For studies on CO signaling, CO-releasing molecules (CORMs) of different structure are available. Here, three frequently used CORMs (CORM-2, CORM-3 and CORM-401) were studied for their properties to provide CO in biological test systems and address susceptible heme proteins. CO release was investigated in the myoglobin binding assay and found to be rapid (<5 min) with CORM-2- and CORM-3, whereas CORM-401 continuously provided CO (>50 min). Storage stability of CORM stock solutions was also assessed with the myoglobin assay. Only CORM-401 stock solutions were stable over a period of 7 days. Incubation of CORMs with recombinant cytochrome P450 led to an inhibition of enzyme activity. However, only CORM-3 and CORM-401 proved to be suitable in this test system because controls with the inactivated CORM-2 (iCORM-2) also led to a loss of enzyme activity. The impact of CORMs on the respiratory chain was investigated with high resolution respirometry and extracellular flux technology. In the first approach interferences of CORM-2 and CORM-3 with oxygen measurement occurred, since a rapid depletion of oxygen was detected in the medium even when no cells were present. However, CORM-401 did not interfere with oxygen measurement and the expected inhibition of cellular respiration was observed. CORM-2 was not suitable for use in oxygen measurements with the extracellular flux technology and CORM-3 application did not show any effect in this system. However, CO-dependent inhibition of cellular respiration was observed with CORM-401. Based on the present experiments it is concluded, that CORM-401 produced most reliable CO-specific results for the modulation of typical CO targets. For studies on CO-dependent biological effects on intracellular heme groups, CORM-2 and CORM-3 were less suitable. Depending on the experimental setting, data achieved with these compounds should be evaluated with caution.
Subject(s)
Carbon Monoxide/metabolism , N-substituted Glycines/pharmacology , Organometallic Compounds/pharmacology , Animals , Cell Respiration/drug effects , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Drug Stability , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Mice , N-substituted Glycines/chemistry , Organometallic Compounds/chemistryABSTRACT
HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.
Subject(s)
Mycobacterium tuberculosis/metabolism , Tuberculosis, Pulmonary/metabolism , Bayes Theorem , Codon , Coinfection/metabolism , Evolution, Molecular , Genetic Variation , HIV Infections/complications , HIV Infections/genetics , HIV-1/genetics , Humans , Models, Genetic , Mutation , Mycobacterium tuberculosis/drug effects , Selection, Genetic , Tuberculosis, Pulmonary/complicationsABSTRACT
Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.
Subject(s)
Antitubercular Agents/therapeutic use , Diarylquinolines/therapeutic use , Drug Resistance, Bacterial/genetics , Mutation , Mycobacterium tuberculosis/genetics , Nitroimidazoles/therapeutic use , Oxazoles/therapeutic use , Tuberculosis, Multidrug-Resistant/drug therapy , Drug Therapy, Combination , Humans , Male , Mycobacterium tuberculosis/drug effectsABSTRACT
Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.
Subject(s)
Disease Transmission, Infectious , Emigrants and Immigrants , Molecular Typing , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/genetics , Tuberculosis/transmission , Adolescent , Adult , Cluster Analysis , Female , Follow-Up Studies , Genome, Bacterial , Humans , Incidence , Male , Middle Aged , Molecular Epidemiology , Mycobacterium tuberculosis/isolation & purification , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Switzerland/epidemiology , Tuberculosis/epidemiology , Tuberculosis/microbiology , Young AdultABSTRACT
The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.
Subject(s)
Adaptation, Physiological , Host-Pathogen Interactions , Mycobacterium tuberculosis , Tuberculosis , Adult , Aged , CD4-Positive T-Lymphocytes , Evolution, Molecular , Female , HIV/pathogenicity , HIV Infections/complications , HIV Infections/genetics , HIV Infections/virology , Humans , Male , Middle Aged , Molecular Epidemiology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/physiology , Phylogeography , Switzerland , Sympatry , Tuberculosis/complications , Tuberculosis/genetics , Tuberculosis/microbiologyABSTRACT
BACKGROUND: Whole-genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single-nucleotide polymorphism (SNP) typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. METHODS: On the basis of genome sequences of 3 historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1642 patient isolates and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. RESULTS: We identified 68 patients associated with the outbreak strain. Most received a tuberculosis diagnosis in 1991-1995, but cases were observed until 2011. Two thirds were homeless and/or substance abusers. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into 3 subclusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. CONCLUSIONS: Strain-specific SNP genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real time and at high resolution.
Subject(s)
Genome, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Polymorphism, Single Nucleotide/genetics , Tuberculosis/epidemiology , Tuberculosis/microbiology , Adult , Aged , Bacterial Typing Techniques/methods , DNA, Bacterial/genetics , Disease Outbreaks , Female , Genotype , Humans , Male , Middle Aged , Molecular Epidemiology/methods , Retrospective Studies , Sequence Analysis, DNA/methods , Switzerland/epidemiologyABSTRACT
BACKGROUND: High-throughput DNA sequencing produces vast amounts of data, with millions of short reads that usually have to be mapped to a reference genome or newly assembled. Both reference-based mapping and de novo assembly are computationally intensive, generating large intermediary data files, and thus require bioinformatics skills that are often lacking in the laboratories producing the data. Moreover, many research and practical applications in microbiology require only a small fraction of the whole genome data. RESULTS: We developed KvarQ, a new tool that directly scans fastq files of bacterial genome sequences for known variants, such as single nucleotide polymorphisms (SNP), bypassing the need of mapping all sequencing reads to a reference genome and de novo assembly. Instead, KvarQ loads "testsuites" that define specific SNPs or short regions of interest in a reference genome, and directly synthesizes the relevant results based on the occurrence of these markers in the fastq files. KvarQ has a versatile command line interface and a graphical user interface. KvarQ currently ships with two "testsuites" for Mycobacterium tuberculosis, but new "testsuites" for other organisms can easily be created and distributed. In this article, we demonstrate how KvarQ can be used to successfully detect all main drug resistance mutations and phylogenetic markers in 880 bacterial whole genome sequences. The average scanning time per genome sequence was two minutes. The variant calls of a subset of these genomes were validated with a standard bioinformatics pipeline and revealed >99% congruency. CONCLUSION: KvarQ is a user-friendly tool that directly extracts relevant information from fastq files. This enables researchers and laboratory technicians with limited bioinformatics expertise to scan and analyze raw sequencing data in a matter of minutes. KvarQ is open-source, and pre-compiled packages with a graphical user interface are available at http://www.swisstph.ch/kvarq.
Subject(s)
Bacteria/genetics , Genome, Bacterial , Software , Algorithms , Bacteria/classification , Drug Resistance, Bacterial/genetics , Internet , Mycobacterium tuberculosis/genetics , Phylogeny , Polymorphism, Single Nucleotide , User-Computer InterfaceABSTRACT
Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.
Subject(s)
Swallows , Animals , Swallows/genetics , Metagenomics , Genome/genetics , Genomics , ChromosomesABSTRACT
Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.
Subject(s)
Emigration and Immigration , HIV Infections/complications , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/epidemiology , Tuberculosis/transmission , Adult , Bacterial Typing Techniques , Cluster Analysis , Cohort Studies , Female , Humans , Incidence , Male , Middle Aged , Molecular Epidemiology , Molecular Typing , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/genetics , Switzerland/epidemiology , Tuberculosis/complicationsABSTRACT
The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.
Subject(s)
Escherichia coli O157/growth & development , Pseudomonas aeruginosa/growth & development , Vibrio cholerae/growth & development , Water Microbiology , Bacterial Load , Carbon/analysis , Flow Cytometry , Organic Chemicals/analysis , Water/analysis , Water PurificationABSTRACT
Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.
Subject(s)
Cerium/pharmacology , Cytotoxins/pharmacology , Melanoma/drug therapy , Mitochondria/drug effects , Antioxidants/chemistry , Antioxidants/pharmacology , Catalase/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cerium/chemistry , Cytotoxins/chemistry , Humans , Melanoma/metabolism , Melanoma/pathology , Mitochondria/pathology , Nanoparticles/chemistry , Neoplasm Metastasis , Sulfhydryl Compounds/metabolismABSTRACT
Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.
ABSTRACT
Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50⯵M CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , N-substituted Glycines/metabolism , Xenobiotics/metabolism , Amitriptyline/metabolism , Carbon Monoxide/metabolism , Hep G2 Cells , Humans , Nortriptyline/metabolismABSTRACT
Malignant melanoma is an aggressive type of cancer and the deadliest form of skin cancer. Even though enormous efforts have been undertaken, in particular the treatment options against the metastasizing form are challenging and the prognosis is generally poor. A novel therapeutical approach is the application of secondary plant constituents occurring in food and food products. Herein, the effect of the dietary chalcone cardamonin, inter alia found in Alpinia species, was tested using human malignant melanoma cells. These data were compared to cardamonin treated normal melanocytes and dermal fibroblasts representing healthy cells. To investigate the impact of cardamonin on tumor and normal cells, it was added to monolayer cell cultures and cytotoxicity, proliferation, tumor invasion, and apoptosis were studied with appropriate cell biological and biochemical methods. Cardamonin treatment resulted in an apoptosis-mediated increase in cytotoxicity towards tumor cells, a decrease in their proliferation rate, and a lowered invasive capacity, whereas the viability of melanocytes and fibroblasts was hardly affected at such concentrations. A selective cytotoxic effect of cardamonin on melanoma cells compared to normal (healthy) cells was shown in vitro. This study along with others highlights that dietary chalcones may be a valuable tool in anticancer therapies which has to be proven in the future in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Chalcones/pharmacology , Cytotoxins/pharmacology , Melanoma/drug therapy , Skin Neoplasms/drug therapy , Blotting, Western , Cell Line, Tumor , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Melanocytes/drug effects , Reactive Oxygen Species/metabolismABSTRACT
BH3 mimetics, such as BH3I-1, act as Bcl-2 antagonists, promote apoptosis and are used in basic research studies on apoptotic signaling and are currently tested as experimental anti-tumor agents. The present study addresses time- and dose-dependent responses of BH3I-1 on apoptosis, cellular stress defense mediated by heme oxygenase-1 (HO-1), and mitochondrial morphology. As expected, treatment of normal human dermal fibroblasts with BH3I-1 induced apoptosis as determined by typical markers including cytochrome c release, loss of procaspase-3, and PARP cleavage. Induction of the cellular stress response marker HO-1 precedes apoptosis induction whereas fragmentation of the mitochondrial network was triggered even more rapidly. No difference in apoptosis induction was found upon depletion of HO-1 by siRNA compared to controls suggesting that apoptosis induction by BH3I-1 is not affected by HO-1. To evaluate the functional interplay between mitochondrial fragmentation and HO-1 induction, murine embryonic fibroblasts lacking the fission factor Drp1 were used. In Drp1 knock out cells, HO-1 levels were low compared to wild type cells, both in untreated controls as well as after BH3I-1 exposure, demonstrating that Drp1 is at least in part required for determining basal and inducible HO-1 levels. Considering the sequence of events, it was shown here that BH3I-1 dependent apoptosis is a rather late event, while effects on mitochondrial morphology and cellular stress response (HO-1 induction) are observed rapidly after exposure of cells to the compound. We propose that BH3I-1 is a valuable tool for studying cellular stress responses as well as mitochondrial dynamics in future studies. Since BH3 mimetics are promising experimental anticancer drugs, our data further imply that additional biological effects such as upregulation of detoxifying systems or changes in mitochondrial dynamics could interfere, in combination therapy, with selective drug toxicity and thus need to be taken into account for drug development.
Subject(s)
Apoptosis/drug effects , Fibroblasts/drug effects , Mitochondria/drug effects , Mitophagy/drug effects , Oxidative Stress/drug effects , Thiazoles/toxicity , Animals , Apoptosis Regulatory Proteins/metabolism , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Dynamins/genetics , Dynamins/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mitochondria/metabolism , Mitochondria/pathology , Molecular Mimicry , Signal Transduction/drug effects , Thiazolidinediones , Time FactorsABSTRACT
The fast evolution of pathogenic viruses has allowed for the development of phylodynamic approaches that extract information about the epidemiological characteristics of viral genomes. Thanks to advances in whole genome sequencing, they can be applied to slowly evolving bacterial pathogens like Mycobacterium tuberculosis. In this study, we investigate and compare the epidemiological dynamics underlying two M. tuberculosis outbreaks using phylodynamic methods. Specifically, we (i) test if the outbreak data sets contain enough genetic variation to estimate short-term evolutionary rates and (ii) reconstruct epidemiological parameters such as the effective reproduction number. The first outbreak occurred in the Swiss city of Bern (1987-2012) and was caused by a drug-susceptible strain belonging to the phylogenetic M. tuberculosis Lineage 4. The second outbreak was caused by a multidrug-resistant (MDR) strain of Lineage 2, imported from the Wat Tham Krabok (WTK) refugee camp in Thailand into California. There is little temporal signal in the Bern data set and moderate temporal signal in the WTK data set. Thanks to its high sampling proportion (90%) the Bern outbreak allows robust estimation of epidemiological parameters despite the poor temporal signal. Conversely, there is much uncertainty in the epidemiological estimates concerning the sparsely sampled (9%) WTK outbreak. Our results suggest that both outbreaks peaked around 1990, although they were only recognized as outbreaks in 1993 (Bern) and 2004 (WTK). Furthermore, individuals were infected for a significantly longer period (around 9 years) in the WTK outbreak than in the Bern outbreak (4-5 years). Our work highlights both the limitations and opportunities of phylodynamic analysis of outbreaks involving slowly evolving pathogens: (i) estimation of the evolutionary rate is difficult on outbreak time scales and (ii) a high sampling proportion allows quantification of the age of the outbreak based on the sampling times, and thus allows for robust estimation of epidemiological parameters.
Subject(s)
Disease Outbreaks , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/epidemiology , Tuberculosis/microbiology , California , Genetic Variation , Genome, Viral , Humans , Mycobacterium tuberculosis/genetics , Phylogeny , Switzerland , Thailand , Tuberculosis/transmissionABSTRACT
BACKGROUND: Combining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB. METHOD: Spoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages. RESULTS: Of the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed. CONCLUSION: According to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.
Subject(s)
Genotype , Minisatellite Repeats , Mycobacterium tuberculosis/genetics , Polymorphism, Single Nucleotide , MadagascarABSTRACT
Zoonotic tuberculosis is a risk for human health, especially when animals are in close contact with humans. Mycobacterium tuberculosis was cultured from several organs, including lung tissue and gastric mucosa, of three captive elephants euthanized in a Swiss zoo. The elephants presented weight loss, weakness and exercise intolerance. Molecular characterization of the M. tuberculosis isolates by spoligotyping revealed an identical profile, suggesting a single source of infection. Multilocus variable-number of tandem-repeat analysis (MLVA) elucidated two divergent populations of bacteria and mixed infection in one elephant, suggesting either different transmission chains or prolonged infection over time. A total of eight M. tuberculosis isolates were subjected to whole-genome sequence (WGS) analysis, confirming a single source of infection and indicating the route of transmission between the three animals. Our findings also show that the methods currently used for epidemiological investigations of M. tuberculosis infections should be carefully applied on isolates from elephants. Moreover the importance of multiple sampling and analysis of within-host mycobacterial clonal populations for investigations of transmission is demonstrated.
Subject(s)
Diagnostic Tests, Routine/veterinary , Disease Outbreaks/veterinary , Minisatellite Repeats , Multilocus Sequence Typing/veterinary , Mycobacterium tuberculosis/genetics , Tuberculosis/diagnosis , Whole Genome Sequencing/veterinary , Animals , Elephants , Mycobacterium tuberculosis/classification , Switzerland/epidemiology , Tuberculosis/epidemiology , Tuberculosis/genetics , Tuberculosis/microbiologyABSTRACT
Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1(154Q/2Q) mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1(154Q/2Q) mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as prevented oxidative stress-induced DNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.