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2. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Hollestelle, Antoinette, et al.
(author)
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No clinical utility of KRAS variant rs61764370 for ovarian or breast cancer
- 2016
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In: Gynecologic Oncology. - : Elsevier BV. - 0090-8258 .- 1095-6859. ; 141:2, s. 386-401
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Journal article (peer-reviewed)abstract
- Objective Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3′ UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370. Methods Centralized genotyping and analysis were performed for 140,012 women enrolled in the Ovarian Cancer Association Consortium (15,357 ovarian cancer patients; 30,816 controls), the Breast Cancer Association Consortium (33,530 breast cancer patients; 37,640 controls), and the Consortium of Modifiers of BRCA1 and BRCA2 (14,765 BRCA1 and 7904 BRCA2 mutation carriers). Results We found no association with risk of ovarian cancer (OR = 0.99, 95% CI 0.94-1.04, p = 0.74) or breast cancer (OR = 0.98, 95% CI 0.94-1.01, p = 0.19) and results were consistent among mutation carriers (BRCA1, ovarian cancer HR = 1.09, 95% CI 0.97-1.23, p = 0.14, breast cancer HR = 1.04, 95% CI 0.97-1.12, p = 0.27; BRCA2, ovarian cancer HR = 0.89, 95% CI 0.71-1.13, p = 0.34, breast cancer HR = 1.06, 95% CI 0.94-1.19, p = 0.35). Null results were also obtained for associations with overall survival following ovarian cancer (HR = 0.94, 95% CI 0.83-1.07, p = 0.38), breast cancer (HR = 0.96, 95% CI 0.87-1.06, p = 0.38), and all other previously-reported associations. Conclusions rs61764370 is not associated with risk of ovarian or breast cancer nor with clinical outcome for patients with these cancers. Therefore, genotyping this variant has no clinical utility related to the prediction or management of these cancers.
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- Osorio, Ana, et al.
(author)
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DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers.
- 2014
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In: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 10:4
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Journal article (peer-reviewed)abstract
- Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7×10-3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8×10-3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.
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- Coronado, Liani, et al.
(author)
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Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy
- 2019
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In: BMC Veterinary Research. - : BioMed Central (BMC). - 1746-6148. ; 15:1
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Journal article (peer-reviewed)abstract
- Background: Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions.Results: Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage.Conclusions: Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.
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6. |
- Coronado, Liani, et al.
(author)
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Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity : implication for epidemiological surveillance in endemic areas
- 2019
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In: Transboundary and Emerging Diseases. - : Blackwell Verlag. - 1865-1674 .- 1865-1682. ; 66:6, s. 2362-2382
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Journal article (peer-reviewed)abstract
- Classical swine fever (CSF), caused by CSF virus (CSFV), is considered one of the most important infectious diseases with devasting consequences for the pig industry. Recent reports describe the emergence of new CSFV-strains resulting from the action of positive selection pressure, due mainly to the bottleneck effect generated by ineffective vaccination. Even though a decrease in the genetic diversity of the positive selected CSFV-strains has been observed by several research groups, there is little information about the effect of this selective force on the virulence degree, antigenicity and pathogenicity of this type of strains. Hence, the aim of the current study was to determine the effect of the positive selection pressure on these three parameters of CSFV-strains, emerged as result of the bottleneck effects induced by unproper vaccination in a CSF-endemic area. Moreover, the effect of the positive selected strains on the epidemiological surveillance system was assessed. By the combination of in vitro, in vivo and immunoinformatic approaches we revealed that the action of the positive selection pressure induces a decrease in virulence and alteration in pathogenicity and antigenicity. However, we also noted that the evolutionary process of CSFV, especially in segregated microenvironments, could contribute to the gain-fitness event, restoring the highly virulent pattern of the circulating strains. Besides, we denoted that the presence of low virulent strains selected by bottleneck effect after inefficient vaccination can lead to a relevant challenge for the epidemiological surveillance of CSF, contributing to under-reports of the disease, favoring the perpetuation of the virus in the field. In this study B-cell and CTL epitopes on the E2 3D-structure model were also identified. Thus, the current study provides novel and significant insights into variation in virulence, pathogenesis and antigenicity experienced by CSFV strains after the positive selection pressure effect.
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7. |
- Hamdi, Yosr, et al.
(author)
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Association of breast cancer risk in BRCA1 and BRCA2 mutation carriers with genetic variants showing differential allelic expression : identification of a modifier of breast cancer risk at locus 11q22.3
- 2017
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In: Breast Cancer Research and Treatment. - : Springer Science and Business Media LLC. - 0167-6806 .- 1573-7217. ; 161:1, s. 117-134
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Journal article (peer-reviewed)abstract
- Purpose: Cis-acting regulatory SNPs resulting in differential allelic expression (DAE) may, in part, explain the underlying phenotypic variation associated with many complex diseases. To investigate whether common variants associated with DAE were involved in breast cancer susceptibility among BRCA1 and BRCA2 mutation carriers, a list of 175 genes was developed based of their involvement in cancer-related pathways. Methods: Using data from a genome-wide map of SNPs associated with allelic expression, we assessed the association of ~320 SNPs located in the vicinity of these genes with breast and ovarian cancer risks in 15,252 BRCA1 and 8211 BRCA2 mutation carriers ascertained from 54 studies participating in the Consortium of Investigators of Modifiers of BRCA1/2. Results: We identified a region on 11q22.3 that is significantly associated with breast cancer risk in BRCA1 mutation carriers (most significant SNP rs228595 p = 7 × 10−6). This association was absent in BRCA2 carriers (p = 0.57). The 11q22.3 region notably encompasses genes such as ACAT1, NPAT, and ATM. Expression quantitative trait loci associations were observed in both normal breast and tumors across this region, namely for ACAT1, ATM, and other genes. In silico analysis revealed some overlap between top risk-associated SNPs and relevant biological features in mammary cell data, which suggests potential functional significance. Conclusion: We identified 11q22.3 as a new modifier locus in BRCA1 carriers. Replication in larger studies using estrogen receptor (ER)-negative or triple-negative (i.e., ER-, progesterone receptor-, and HER2-negative) cases could therefore be helpful to confirm the association of this locus with breast cancer risk.
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8. |
- Lawitz, Eric, et al.
(author)
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Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY) : a randomised, open-label phase 2 trial
- 2015
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In: The Lancet. - 0140-6736 .- 1474-547X. ; 385:9973, s. 1075-1086
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Journal article (peer-reviewed)abstract
- BACKGROUND: There is a high medical need for an interferon-free, all-oral, short-duration therapy for hepatitis C virus (HCV) that is highly effective across diverse patient populations, including patients with cirrhosis or previous null response to pegylated interferon (peginterferon) plus ribavirin (PR-null responders). We aimed to assess the efficacy, safety, and effective treatment duration of grazoprevir (an HCV NS3/4A protease inhibitor) combined with elbasvir (an HCV NS5A inhibitor) with or without ribavirin in patients with HCV genotype 1 infection with baseline characteristics of poor response.METHODS: The C-WORTHY trial is a randomised, open-label phase 2 trial of grazoprevir plus elbasvir with or without ribavirin; here we report findings for two cohorts of previously untreated patients with cirrhosis (cohort 1) and those with previous PR-null response with or without cirrhosis (cohort 2) enrolled in part B of the study. Eligible patients were adults aged 18 years or older with chronic HCV genotype 1 infection and HCV RNA concentrations of 10 000 IU/mL or higher in peripheral blood. We randomly assigned patients to receive grazoprevir (100 mg daily) and elbasvir (50 mg daily) with or without ribavirin for 12 or 18 weeks. Randomisation was done centrally with an interactive voice response system; patients and study investigators were masked to treatment duration up to week 12 but not to treatment allocation. The primary endpoint was the proportion of patients achieving HCV RNA less than 25 IU/mL at 12 weeks after end of treatment (SVR12), assessed by COBAS TaqMan version 2.0. This study is registered with ClinicalTrials.gov, number NCT01717326.FINDINGS: We describe findings for 253 patients enrolled in cohort 1 (n=123) or cohort 2 (n=130). In cohort 1, we randomly assigned 60 patients to the 12-week regimen (31 with ribavirin and 29 with no ribavirin) and 63 to the 18-week regimen (32 with ribavirin and 31 with no ribavirin); in cohort 2, we randomly assigned 65 patients to the 12-week regimen (32 with ribavirin and 33 with no ribavirin) and 65 to the 18-week regimen (33 with ribavirin and 32 with no ribavirin. High SVR12 rates were achieved irrespective of the use of ribavirin or extension of the treatment duration from 12 to 18 weeks; SVR12 rates ranged from 90% (95% CI 74-98; 28/31; cohort 1, 12 weeks, ribavirin-containing) to 100% (95% CI 89-100; 33/33; cohort 2, 18 weeks, ribavirin-containing). Among patients treated for 12 weeks with grazoprevir plus elbasvir without ribavirin, 97% (95% CI 82-100, 28/29) of patients in cohort 1 and 91% (76-98, 30/33) of patients in cohort 2 achieved SVR12. Adverse events reported in more than 10% of patients were fatigue (66 patients, 26% [95% CI 21-32]), headache (58 patients, 23% [95% CI 18-29]), and asthenia (35 patients, 14% [95% CI 10-19]).INTERPRETATION: Treatment with grazoprevir plus elbasvir, both with and without ribavirin and for both 12 and 18 weeks' treatment duration, showed high rates of efficacy in previously untreated patients with cirrhosis and previous PR-null responders with and without cirrhosis. These results support the phase 3 development of grazoprevir plus elbasvir.FUNDING: Merck & Co, Inc.
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- Lawrenson, Kate, et al.
(author)
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Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus
- 2016
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Journal article (peer-reviewed)abstract
- A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk.
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- Lindblad-Toh, Kerstin, et al.
(author)
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Genome sequence, comparative analysis and haplotype structure of the domestic dog.
- 2005
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In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 438:7069, s. 803-19
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Journal article (peer-reviewed)abstract
- Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.
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