The in vivo characterization of the DNA repair gene apn-1 in the model organism Caenorhabditis elegans

Les sites apuriniques/apyrimidinique (AP) représentent une forme de dommage à l’ADN hautement mutagène et ce type de dommage peut survenir spontanément ou être induit par une variété d’agents. Afin de préserver la stabilité génomique, deux familles d’endonucléases de type AP, endo-IV et exo-III, sont nécessaires pour contrecarrer les effets mutagènes des sites AP. Malgré l’identification de membres des deux familles dans plusieurs organismes unicellulaire tels que E.coli et S. cerevisiae, aucun membre de la famille endo-IV n’a été identifié chez les organismes multicellulaires à l’exception de C. elegans et de C. briggsae. Nous avons donc décidé d’investiguer l’importance biologique de APN-1 chez C. elegans par l’utilisation d’une approche de knockdown du gène. Dans notre étude, nous avons montré que le knockdown du gène apn-1 chez C. elegans, en utilisant des ARN d’interférence (ARNi), cause une accumulation de mutations spontanées et induites par des drogues résultant en un délai de l’éclosion des œufs ainsi que par une diminution de la survie et de la longévité des vers adultes. De plus, nous avons montré que cette accumulation de mutations mène à un délai dans la progression du cycle cellulaire durant l’embryogénèse, représentant possiblement une explication du délai dans l’éclosion des œufs. Nous avons montré qu’il y avait une augmentation du niveau de mutations dans la gorge des vers, sans toutefois pouvoir confirmer la distribution de APN-1 qui possède une étiquette GFP. Les animaux transgéniques APN-1-GFP n’exprimaient pas suffisamment de la protéine de fusion pour permettre une visualisation à l’aide d’un microscope à fluorescence, mais la protéine a été détectée par immunobuvardage de type western. Les animaux transgéniques APN-1-GFP étaient instables et avaient des phénotypes concordants avec les défauts génétiques. En conclusion, il semble que C. elegans aie évolué afin de retenir un niveau de base de APN-1 jouant ainsi un rôle versatile afin de maintenir l’intégrité génétique d’autant plus que cet organisme semble manquer plusieurs enzymes de la voie de réparation par excision de base.Apurinic/apyrimidinic (AP) sites are a form of highly mutagenic DNA damage that arise either spontaneously or by a variety of DNA damaging agents. To preserve genomic stability two AP endonuclease families, endo-IV and exo-III, evolved to counteract the mutagenic effect of AP sites. While members of both families were identified in multiple unicellular organisms, notably E. coli and S. cerevisiae, no members of the endo-IV family were identified in multicellular ones, with the exception of C. elegans and its close relatives, particularly C. briggsae. We set out to investigate the biological importance of APN-1 in C. elegans using gene knockdown approach. In our study, we showed that the knockdown of C. elegans apn-1 gene, using RNAi causes the accumulation of spontaneous and drug induced mutations, resulting in a delay in egg hatching, decreased survival and longevity. Furthermore, we have showed that the accumulated mutations lead to delays in cell cycle progression during early embryogenesis, thus providing a possible explanation for the observed delay in hatching. Although we showed increased mutations in the gut of the worm, we were unable to confirm APN-1 distribution tagged with GFP. The transgenic APN-1-GFP animal did not express enough of this fusion protein to be visualized by fluorescent microscopy, although it was detected by Western blot analysis. The transgenic animals over-expressing APN-1-GFP were unstable and showed phenotypes consistent with genetic defects. In conclusion, it would seem that C. elegans has evolved to retain a balanced level of APN-1, which plays a versatile role in maintaining genetic integrity, since this organism lacks a full complement of the enzymes in the base-excision repair pathway

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway

Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.</div

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Exploring the effects and effectors of mTOR inhibition in oncolysis and TOP mRNA translation

Translation is a fundamental, energy-consuming, process that permits quick and accurate responses to cellular and environmental conditions. The mammalian target of rapamycin complex 1 (mTORC1) acts as a central hub relaying signals to the translation machinery, which results in the modulation of various stages of mRNA translation. mTORC1 pathway is dysregulated in many human malignancies. As such, small molecule mTOR inhibitors are being investigated for cancer treatment either alone or in combination with other anti-cancer therapies. Different subsets of mRNAs are regulated in an mTORC1-dependent manner. These include transcripts encoding for some components of the innate antiviral response, characterized by long and complex 5'-UTR, as well as terminal oligopyrimidine (TOP) mRNAs, encoding for components of the translational machinery. The best characterized mTORC1 effectors are the ribosomal protein S6 kinases (S6Ks) and eIF4E-binding proteins (4E-BPs). While many transcripts encoding for antiviral response are highly regulated by mTORC1 via 4E-BPs, the identity of the mTORC1 effector in TOP regulation remains unknown. In chapter 2 of this thesis, we report the specific potentiation of oncolytic HSV1 infection by active site mTOR inhibitors (asTORi) in transformed cells, while recapitulating the previously published antiviral effects of asTORi in non-transformed cells. We further demonstrate that this differential response to asTORi is dependent on the eIF4E/4E-BPs ratio, whereby a high ratio results in specific translational suppression of the antiviral response, while a low ratio inhibits the translation of the antiviral response and the virus. In chapter 3 of this thesis, we report LARP1 as a novel mTORC1 substrate mediating TOP mRNA repression and stability. Our data show enhanced LARP1 binding to TOP mRNAs when mTOR is inhibited, and reveal a LARP1 dependency for TOP mRNA repression by mTOR inhibitors. Finally, we demonstrate direct competition between LARP1 and eIF4G for TOP mRNA binding, suggesting that LARP1 prevents eIF4F complex assembly under conditions of mTOR inhibition. In conclusion, understanding the functions and implications of the diverse mTORC1 effectors in malignant transformation is essential for predicting therapeutic outcome of mTOR inhibition and lays the foundation for the assessment of combinational therapies.La traduction est un processus fondamental hautement énergivore qui permet des réponses rapides et précises aux conditions cellulaires et environnementales. Le mammalian target of rapamycin complex 1 (mTORC1) agit comme principal régulateur de la traduction d'ARNm. La voie de signalisation dont mTORC1 fait partie étant dérégulée dans plusieurs maladies humaines, de nombreux inhibiteurs ciblant mTOR ont été développés. Ceux-ci sont efficaces dans le traitement de certains cancers, utilisés seuls ou en combinaison avec d'autres thérapies anti-cancer. Différents groupes d'ARNm sont régulés par mTORC1. Ceux-ci incluent des transcrits encodant certains acteurs de la réponse antivirale innée, caractérisés par des régions non-traduites en 5' (5' UTR) longues et complexes, ainsi que les ARNm TOP (terminal oligopyrimidine), encodant divers composés de la machinerie de traduction. Les effecteurs de mTORC1 les mieux caractérisés sont les ribosomal protein S6 kinases (S6Ks) et les eIF4E-binding proteins (4E-BPs). Bien que plusieurs facteurs impliqués dans la réponse antivirale soient fortement régulés par mTORC1 via les 4E-BPs, l'identité de l'effecteur de mTORC1 impliqué dans la régulation des ARNm TOP est toujours inconnue. Dans le chapitre 2 de cette thèse, nous reportons la potentialisation spécifique de l'infection oncolytique HSV1 par des inhibiteurs du site actif de mTOR (asTORi) dans les cellules transformées, tout en récapitulant les résultats publiés précédemment des effets antiviraux des asTORi dans les cellules non-transformées. De plus, nous démontrons que cette réponse différentielle aux inhibiteurs asTORi est dépendante du ratio eIF4E/4E-BPs, par lequel un haut ratio résulte en la suppression spécifique de la traduction de la réponse antivirale, tandis qu'un bas ratio inhibe le virus. Dans le chapitre 3 de cette thèse, nous identifions LARP1 en tant que nouveau substrat de mTORC1, responsable de son effet sur la répression et la stabilité des ARNm TOP. Nos données démontrent une augmentation de la liaison de LARP1 aux ARNm TOP lorsque mTOR est inhibé, et révèle la dépendance des inhibiteurs de mTOR à l'activité de LARP1 pour la répression d'ARNm TOP. Finalement, nous démontrons qu'il y a une compétition directe entre LARP1 et eIF4G pour la liaison aux ARNm TOP, suggérant que LARP1 prévient l'assemblage du complexe eIF4F dans des conditions où mTOR est inhibé. En conclusion, les travaux inclus dans cette thèse permettent de mieux comprendre les fonctions de divers effecteurs de mTORC1 dans les cellules cancéreuses, permettent de mieux prédire le succès thérapeutique d'inhibiteurs de mTOR, et posent les fondements de leur éventuelle utilité dans des thérapies combinatoires avec des virus oncolytiques

Objective Sustainability Assessment in the Digital Economy: An Information Entropy Measure of Transparency in Corporate Sustainability Reporting

The Internet is now a central enabler for sharing sustainability information. Yet, such enablement is complicated through an exponentially increasing array of information. What is lacking in the digital economy are objective and transparent mechanisms to provide reliable assessments of the published sustainability information in a timely and efficient manner. In addressing such limitation, this research proposes an objective automated mechanism for measuring transparency in sustainability reporting using an information entropy-based approach. Through text-mining methods and expert validation, the study built a sustainability dictionary corpus and then applied the corpus for objectively assessing the relative entropy between the probability distributions of words in the sustainability dictionary and those in corporate reports. To demonstrate its effectiveness, the mechanism was empirically applied to compare sustainability reporting of organizations in the energy sector. Here, the research effectively compared cartels with non-cartels by assessing the sustainability reports of major OPEC (Organization of the Petroleum Exporting Countries) and non-OPEC producers spanning a three-year period and found consistent differences in transparency between the two groups. The findings demonstrate likely normative transparency pressures on disaffiliated producers for which cartels may be immune. The automated mechanism holds important theoretical and practical contributions to the field of sustainability as it provides a rapid and objective means for textual analysis of sustainability information, thus promoting transparency in sustainability reporting in the rapidly evolving digital economy

Objective Sustainability Assessment in the Digital Economy: An Information Entropy Measure of Transparency in Corporate Sustainability Reporting

The Internet is now a central enabler for sharing sustainability information. Yet, such enablement is complicated through an exponentially increasing array of information. What is lacking in the digital economy are objective and transparent mechanisms to provide reliable assessments of the published sustainability information in a timely and efficient manner. In addressing such limitation, this research proposes an objective automated mechanism for measuring transparency in sustainability reporting using an information entropy-based approach. Through text-mining methods and expert validation, the study built a sustainability dictionary corpus and then applied the corpus for objectively assessing the relative entropy between the probability distributions of words in the sustainability dictionary and those in corporate reports. To demonstrate its effectiveness, the mechanism was empirically applied to compare sustainability reporting of organizations in the energy sector. Here, the research effectively compared cartels with non-cartels by assessing the sustainability reports of major OPEC (Organization of the Petroleum Exporting Countries) and non-OPEC producers spanning a three-year period and found consistent differences in transparency between the two groups. The findings demonstrate likely normative transparency pressures on disaffiliated producers for which cartels may be immune. The automated mechanism holds important theoretical and practical contributions to the field of sustainability as it provides a rapid and objective means for textual analysis of sustainability information, thus promoting transparency in sustainability reporting in the rapidly evolving digital economy

Measuring Corporate Transparency in Sustainability Reporting

Corporations play a central role in sustainability, as their operations have major ramifications on the ecological, social, and economic contexts in which they operate. These operations need to be transparent to all stakeholders so that meaningful reporting could be easily accessed and understood by stakeholders. Indeed, transparency is an important measure of the effectiveness of corporate sustainability reporting (Kaptein et al., 2003). Bushman et al. (2003) defined transparency as the availability of firm-related information to the public, which is external to the organization. The most influential media outlets for firms to report their sustainability efforts include annual reports, social media, and websites. While several researchers have studied measures for corporate transparency (e.g. Delmas et al., 2010; Eldomiaty, 2005), broad-based quantitative approaches grounded in automated text-analysis have not yet attracted much attention. The key objective of this research is to develop a measure of corporate transparency in sustainability reporting and a related methodological approach to examining the sustainability reporting practices of organizations. \ \ The first step in the proposed research is to build a dictionary of keywords in a systematic way through text analytics of a corpus of sustainability-rich documents compiled from different sources such as seminal articles, authoritative subject-specific books, social media, websites, and sustainability-specific reports of companies. The quality of the corpus is then evaluated both analytically and with the help of subject experts. The probability distribution of terms in the sustainability dictionary relative to the original corpus documents, will serve as a basis for developing an entropy-based measure of transparency in the sustainability reporting of businesses (Cover and Thomas, 1991). More specifically, we define a transparency score of a corporate report as the cross entropy between the probability distribution of sustainability terms in the dictionary and sustainability terms in a corporate report. Besides helping to compare reporting across groups of organizations within a specific industry sector, the measure will also help determine which information media are effective indicators of corporate transparency and which are not. The broader aim of this research is to influence improvements in transparency in sustainability reports and in communicating related information to stakeholders, in turn enhancing a corporation’s sustainability strategies and related progress. \ \ Acknowledgement: \ This research was made possible by the generous support of the Qatar Foundation through Carnegie Mellon University in Qatar’s Seed Research program. The statements made herein are solely the responsibility of the author(s).

Metformin requires 4E-BPs to induce apoptosis and repress translation of Mcl-1 in hepatocellular carcinoma cells.

International audienceMetformin inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which is frequently upregulated in hepatocellular carcinoma (HCC). Metformin has also been shown to induce apoptosis in this cancer. Here, we investigate whether metformin-induced apoptosis in HCC is mediated by the downstream mTORC1 effectors eukaryotic initiation factor 4E and (eIF4E)-binding proteins (4E-BPs). Further, we ask whether changes in 4E-BPs activity during metformin treatment negatively regulate translation of the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) mRNA. A genetic HCC mouse model was employed to assess the ability of metformin to reduce tumor formation, induce apoptosis, and control 4E-BP1 activation and Mcl-1 protein expression. In parallel, the HCC cell line Huh7 was transduced with scrambled shRNA (control) or shRNAs targeting 4E-BP1 and 4E-BP2 (4E-BP knock-down (KD)) to measure differences in mRNA translation, apoptosis, and Mcl-1 protein expression after metformin treatment. In addition, immunohistochemical staining of eIF4E and 4E-BP1 protein levels was addressed in a HCC patient tissue microarray. We found that metformin decreased HCC tumor burden, and tumor tissues showed elevated apoptosis with reduced Mcl-1 and phosphorylated 4E-BP1 protein levels. In control but not 4E-BP KD Huh7 cells, metformin induced apoptosis and repressed Mcl-1 mRNA translation and protein levels. Immunostaining of HCC patient tumor tissues revealed a varying ratio of eIF4E/4E-BP1 expression. Our results propose that metformin induces apoptosis in mouse and cellular models of HCC through activation of 4E-BPs, thus tumors with elevated expression of 4E-BPs may display improved clinical chemopreventive benefit of metformin

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway