Institut de Recherche en Infectiologie de Montpellier

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.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. An unbalanced immune response, characterized by a weak production of type I interferons (IFN-Is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. SARS-CoV-2 is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2003 and 2013, respectively. Although IFN treatment gave some encouraging results against SARS-CoV and MERS-CoV in animal models, its potential as a therapeutic against COVID-19 awaits validation. Here, we describe our current knowledge of the complex interplay between SARS-CoV-2 infection and the IFN system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. In addition to the conserved IFN evasion strategies that are likely shared with SARS-CoV and MERS-CoV, novel counteraction mechanisms are being discovered in SARS-CoV-2-infected cells. Since the last coronavirus epidemic, we have made considerable progress in understanding the IFN-I response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pDCs), which are the main IFN-I-producing cells. While awaiting the results of the many clinical trials that are evaluating the efficacy of IFN-I alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed IFN-I treatment and propose strategies to boost pDC-mediated IFN responses during the early stages of viral infection.

A rich body of knowledge links biodiversity to ecosystem functioning (BEF), but it is primarily focused on small scales. We review the current theory and identify six expectations for scale dependence in the BEF relationship: (1) a nonlinear change in the slope of the BEF relationship with spatial scale; (2) a scale-dependent relationship between ecosystem stability and spatial extent; (3) coexistence within and among sites will result in a positive BEF relationship at larger scales; (4) temporal autocorrelation in environmental variability affects species turnover and thus the change in BEF slope with scale; (5) connectivity in metacommunities generates nonlinear BEF and stability relationships by affecting population synchrony at local and regional scales; (6) spatial scaling in food web structure and diversity will generate scale dependence in ecosystem functioning. We suggest directions for synthesis that combine approaches in metaecosystem and metacommunity ecology and integrate cross-scale feedbacks. Tests of this theory may combine remote sensing with a generation of networked experiments that assess effects at multiple scales. We also show how anthropogenic land cover change may alter the scaling of the BEF relationship. New research on the role of scale in BEF will guide policy linking the goals of managing biodiversity and ecosystems.

The fully active dihydroxylated metabolite of vitamin D3 induces the expression ofCYP3A4 and, to a lesser extent, CYP2B6 andCYP2C9 genes in normal differentiated primary human hepatocytes. Electrophoretic mobility shift assays and cotransfection in HepG2 cells using wild-type and mutated oligonucleotides revealed that the vitamin D receptor (VDR) binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP3A4, CYP2B6, andCYP2C9 promoters and shown to be targeted by the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR). Full VDR response of various CYP3A4 heterologous/homologous promoter-reporter constructs requires both the proximal ER6 and the distal DR3 motifs, as observed previously with rifampicin-activated PXR. Cotransfection of a CYP3A4 homologous promoter-reporter construct (including distal and proximal PXR-binding motifs) and of PXR or CAR expression vectors in HepG2 cells revealed the ability of these receptors to compete with VDR for transcriptional regulation of CYP3A4. In conclusion, this work suggests that VDR, PXR, and CAR control the basal and inducible expression of several CYP genes through competitive interaction with the same battery of responsive elements. The fully active dihydroxylated metabolite of vitamin D3 induces the expression ofCYP3A4 and, to a lesser extent, CYP2B6 andCYP2C9 genes in normal differentiated primary human hepatocytes. Electrophoretic mobility shift assays and cotransfection in HepG2 cells using wild-type and mutated oligonucleotides revealed that the vitamin D receptor (VDR) binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP3A4, CYP2B6, andCYP2C9 promoters and shown to be targeted by the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR). Full VDR response of various CYP3A4 heterologous/homologous promoter-reporter constructs requires both the proximal ER6 and the distal DR3 motifs, as observed previously with rifampicin-activated PXR. Cotransfection of a CYP3A4 homologous promoter-reporter construct (including distal and proximal PXR-binding motifs) and of PXR or CAR expression vectors in HepG2 cells revealed the ability of these receptors to compete with VDR for transcriptional regulation of CYP3A4. In conclusion, this work suggests that VDR, PXR, and CAR control the basal and inducible expression of several CYP genes through competitive interaction with the same battery of responsive elements. cytochrome P450 everted repeat (prefixes “p” and “d” indicate proximal and distal, respectively) direct repeat nuclear receptor distal pregnane X-responsive element 25-(OH)2D3, 1α,25-dihydroxyvitamin D3 vitamin D-responsive element reverse transcription glyceraldehyde-3-phosphate dehydrogenase rat atrial natriuretic factor. The official nomenclature system for the nuclear receptor superfamily has been used in this work (1Nuclear Receptor National Committee Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar): PXR, pregnane X receptor (NR1I2) constitutive androstane receptor (NR1I3) retinoid X receptor (NR2B1) vitamin D receptor (NR1I1) Cytochrome P450 (CYP)1enzymes are mainly expressed in the liver and catalyze the metabolic conversion of xenobiotics, including environmental pollutants and drugs, to more polar and easily disposable derivatives (2Denison M.S. Whitlock J.P., Jr. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 3Gonzalez F.J. Trends Pharmacol. Sci. 1992; 13: 346-352Abstract Full Text PDF PubMed Scopus (392) Google Scholar).CYP genes from the CYP2 and CYP3families are inducible by many xenobiotics, notably including barbiturates and rifampicin. Two nuclear receptors, the pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3), have recently been shown to mediate CYP2 andCYP3 gene induction in animals and man (4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 5Waxman D.J. Arch. Biochem. Biophys. 1999; 369: 11-23Crossref PubMed Scopus (670) Google Scholar, 6Zelko I. Negishi M. Biochem. Biophys. Res. Commun. 2000; 277: 1-6Crossref PubMed Scopus (108) Google Scholar). Both PXR and CAR form heterodimers with the retinoid X receptor (RXR; NR2B1). PXR is activated by a wide spectrum of xenobiotics and steroids (4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 7Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow B.M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (796) Google Scholar,8Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar) and controls CYP3A4 and CYP3A7 induction by targeting two specific responsive elements present in the regulatory region of these genes (4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 7Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow B.M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (796) Google Scholar, 8Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar, 9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar, 10Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 11Pascussi J.-M. Jounaidi Y. Drocourt L. Domergue J. Balabaud C. Maurel P. Vilarem M.-J. Biochem. Biophys. Res. Commun. 1999; 260: 377-381Crossref PubMed Scopus (117) Google Scholar, 12Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). The first of these is the proximal PXR-responsive element, located at –160. It consists of an everted repeat of the nuclear receptor half-site AGGTCA separated by 6 nucleotides (ER6); this element is necessary but not sufficient for full transactivation of the CYP3A4 promoter. Indeed, full PXR-mediated induction requires the presence of a second distal xenobiotic-responsive element (dPXRE), located between –7800 and −7200 (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar). This element is composite and consists of two direct repeats separated by 3 nucleotides (DR3), encompassing an ER6 motif. In contrast to PXR, CAR is sequestered in the cytoplasm and translocates into the nucleus upon activation, notably in response to phenobarbital (6Zelko I. Negishi M. Biochem. Biophys. Res. Commun. 2000; 277: 1-6Crossref PubMed Scopus (108) Google Scholar, 13Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar). Several groups have identified a complex phenobarbital-responsive element module that consists of two nuclear receptor-binding sites (termed NR1 and NR2) and one nuclear factor 1 binding site (12Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 14Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst.). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). Both NR1 and NR2 are imperfect DR4 motifs and essential for phenobarbital induction of In human CYP2B6, the phenobarbital-responsive element module is located between and and has been shown to to and be by CAR and by PXR (12Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, B. Moore L.B. Stoltz McKee D.D. Kliewer S.A. Mol. Pharmacol. Google Scholar). revealed that 1α,25-dihydroxyvitamin the active metabolite of vitamin as a transcriptional ofCYP3A4 in the and in the human P. J.M. Mol. Pharmacol. PubMed Scopus Google Scholar, C. K. J. T. Y. H. E. J. E. Mol. Pharmacol. PubMed Scopus Google Scholar). is through the vitamin D receptor binding with heterodimers with C. J. Biochem. 1995; PubMed Scopus Google Scholar, C. J. Invest. Proc. Google Scholar, C. J. Invest. Proc. Google Scholar). The binds to and transactivates the vitamin D elements present in the regulatory region of genes S. J. Biochem. 2000; PubMed Scopus Google Scholar). The of a direct repeat of nuclear receptor separated by 3 nucleotides C. J. Biochem. 1995; PubMed Scopus Google Scholar). In the vitamin D-responsive including the and vitamin VDR a in the regulation of and and of and A. E. J. 1999; 277: PubMed Google Scholar). VDR is expressed in many vitamin D-responsive including the and and in cells U. P. K. J. Clin. PubMed Scopus Google in controls a of including of and and S. T. 2000; Google Scholar, S. Y. M. PubMed Scopus Google Scholar, 1992; 13: PubMed Google Scholar). In an of this that is an ofCYP3A4 in human as previously observed by in P. J.M. Mol. Pharmacol. PubMed Scopus Google Scholar, C. K. J. T. Y. H. E. J. E. Mol. Pharmacol. PubMed Scopus Google Scholar). that VDR be to and/or elements the is be an of CYP genes by these The that not CYP3A4, but CYP2B6 andCYP2C9 in primary human hepatocytes. In that VDR is to and motifs by PXR and CAR in the promoters of and from from and from The expression by of of human PXR by S. using oligonucleotides and and into The CAR expression by M. Negishi The CYP3A4 the proximal ER6 element by from a previously Y. Maurel P. Vilarem M.-J. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) used as a and from oligonucleotides that sites for of the and of the This into of a gene to the homologous construct and by the or region of CYP3A4 (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google by from human into with and as for the homologous that the in of the gene by the promoter. by of the S. M. J.-M. L. Vilarem M.-J. Maurel P. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google Scholar) of a gene by the in the by of the NR1 of (12Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar) of a gene by the in the and by of the of (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar) of a gene by the in the HepG2 cells from the of in with and of in with 6 as by the using of and of expression by P. used of and of expression Cotransfection of human PXR or CAR using and of both expression and to the of an control of of used in the and or in of and for and as J.-M. Jounaidi Y. Drocourt L. Domergue J. Balabaud C. Maurel P. Vilarem M.-J. Biochem. Biophys. Res. Commun. 1999; 260: 377-381Crossref PubMed Scopus (117) Google Scholar). from liver from for to from liver in this an a the liver not of the presence of a a liver for of a and a liver for of a and to the previously L. I. G. Domergue J. G. Maurel P. Google Scholar, L. I. M. Domergue J. H. Maurel P. Mol. Pharmacol. 1992; Google Scholar). The cells with at in a of 6 of a and of a of and by cells in the presence of the of for an and using to the transcription with 1 of using the reverse to the of the to of CYP3A4, CYP2B6, and using the CYP3A4, CYP2B6, and the at for and of of at for at for 6 and at for In the of the by a the same for the at and reverse as CYP3A4, and and CYP2B6, and and and and K. K. Y. Y. A. M. M. J. Biochem. 2000; PubMed Scopus Google Scholar). Electrophoretic mobility shift assays using VDR and by in using a system for at with of oligonucleotides in 1 1 The to a in and 1 The oligonucleotides used as or as is with in and rat atrial natriuretic factor A. P. C. Biochem. J. 2000; PubMed Scopus Google Scholar). used for the by the to with of in with for CYP by both and using the In a of that as to in this the expression of the a gene to be through VDR K. K. Y. Y. A. M. M. J. Biochem. 2000; PubMed Scopus Google in response to of The are shown in of as by as of the same revealed that a and of CYP3A4 and a of CYP2B6 and the at 1 used to the induction in cells with control from the of from liver as for CYP3A4 1 for CYP2B6 and 1 for In induction for CYP3A4 3 for CYP2B6 and for This gene recently shown to be by and phenobarbital through S. M. J.-M. L. Vilarem M.-J. Maurel P. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.-M. L. M. J.M. Maurel P. Google Scholar). used as controls of not by The that CYP the a vitamin D3 of the is a DR3 this nuclear receptor has been shown to motifs, including and A. P. C. Biochem. J. 2000; PubMed Scopus Google C. of the D R. M. Scholar). that CYP2B6, and CYP3A4 induction by be by VDR through the previously identified and elements. The PXR-responsive elements of CYP3A4 of to as and a distal nuclear receptor motifs, to as and elements to and identified by Goodwin (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google and have been to be the elements mobility shift assays to VDR with these elements. the binding of the in to a by shift as shown in A. a observed both VDR and with the but not these receptors and a an of the not In the specific complex in a in the presence of a or of and or This suggests that these elements be targeted by the In an of the not of the complex 6 and In the of used the same to the binding of VDR to both the and complex observed the with VDR or and and In with the in 3 a observed 3 or 3 in the presence of the and a of the and The of the interaction by using and of including and 6 and and and the phenobarbital-responsive element has been shown to be necessary and sufficient for phenobarbital induction of the gene P. Moore R. J. Negishi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Negishi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). of phenobarbital-responsive elements revealed the presence of two imperfect DR4 motifs and NR2) that to be essential for a full response to In the human CYP2B6 these elements are in with to those in the and rat genes and are located in the region (12Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). are to as and identified a element in the region of human S. M. J.-M. L. Vilarem M.-J. Maurel P. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google Scholar). revealed the presence of an imperfect DR4 to as This element shown to to and be by CAR as as by PXR, to a shown in the both the and motifs as by mobility shift This binding observed in the presence of the and the of the interaction by using and of and that the binding of the to to be of with the binding to the CYP3A4 elements. In these that the binds to the elements of CYP3A4, CYP2B6, of the PXR-responsive elements ofCYP3A4 to to the by VDR by assays in HepG2 with the various and homologous promoter-reporter and with the VDR expression or the expression as a with of for and gene The are shown in the transcriptional of the constructs and of and respectively) in a at 1 This observed in cells with the VDR expression In the of VDR, the transcriptional of these elements by of and VDR expression in HepG2 that the transcriptional of the mutated element or of or the not with homologous promoter-reporter constructs with the of the of transcriptional of these constructs in response to VDR with that observed in response to PXR. this the region the and of the region ofCYP3A4 the in of the gene This construct construct has been shown to be fully responsive to PXR (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google and this in this work Several of this construct and in and transcriptional in response to The are in C. VDR of construct in a In constructs a transcriptional The of in a of transcriptional the proximal by VDR of construct the proximal of by a to the of the transcriptional of and of of that with construct a interaction between the region and the element, as previously for PXR-mediated transactivation of these elements (9Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar). in control PXR CAR activated by not In these that both the proximal region and the distal the motifs are necessary to full VDR response and in the of homologous transactivation by VDR transactivation by of PXR and CAR the transactivation of the CYP3A4 homologous by HepG2 cells with of construct various of PXR or and vectors as The of PXR or CAR from to with the of expression by of of vectors (PXR) or the of VDR expression in for of and to of PXR in the or presence of 1 or 1 The are as induction of in vitamin D or cells to in cells and are the of from two of CAR in the or presence of 1 or The are as of to to CAR basal transactivation and are the of from two with the elements identified in CYP2B6 The are shown in but and of both and constructs observed in the presence of Indeed, transactivation of the CYP3A4 responsive elements the observed This is with the in primary the induction of CYP3A4 in response to is that of both CYP2B6 and 1 VDR binds and transactivates and the of to PXR and CAR compete with this in in HepG2 cells in the presence of a of VDR expression and in the or presence of of PXR or CAR expression vectors or for in the or presence of 1 or a of both or 1 CAR L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (749) Google or a of and gene the are shown in A. The induction are in gene observed in cells in the of transactivation observed in the of this the of receptors in HepG2 VDR not activated by and PXR not activated by of the construct by the in the presence of 1 from to as observed with PXR as the of PXR In in the same transactivation of the construct in the presence of from to as observed with PXR as the of PXR cells with both and transactivation of the construct from a vitamin response to a from observed with VDR to observed with PXR a between PXR and VDR for the CYP3A4 elements. the are shown in B. The are as to to the in basal transactivation of the construct as the of CAR in the of This the that CAR is active in as the of CAR as previously L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). In the that the transcriptional of VDR and CAR not by and of the construct by the in the presence of 1 from to as observed with CAR as the of CAR cells with both and transactivation of the construct from a vitamin response to a the observed with VDR to the observed with CAR in the presence of The for this is in the presence of CAR is the is but is to to responsive a between CAR and VDR for elements. these are in with the shift that VDR to PXR-responsive and elements and in the of the the sites targeted by VDR with those by PXR and In this have shown that induces the expression of gene in normal differentiated primary human and, to a lesser extent, CYP2B6 from mobility shift cotransfection with various oligonucleotides and heterologous/homologous promoter-reporter and between nuclear receptors that VDR is for this induction by those responsive elements previously identified in the promoters of these genes and shown to be targeted by PXR and/or CAR in response to D and is a that requires a for full activation, including a first at mainly in the liver through and a second at mainly in the through A. E. J. 1999; 277: PubMed Google Scholar). This to the of the active form of vitamin This metabolite is mainly in the through at by as as by the of a A. E. J. 1999; 277: PubMed Google Scholar). of vitamin not be or in the are in this cells with a of of the in the normal in the that VDR be or expressed at in the recently that this receptor is present in and rat liver by and C. M. C. T. C. R. Biol. 1999; PubMed Scopus Google Scholar). using the inducible expression of previously shown to be by VDR K. K. Y. Y. A. M. M. J. Biochem. 2000; PubMed Scopus Google have shown that VDR expressed and activated in with nuclear receptor binds to a specific (1Nuclear Receptor National Committee Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, D.J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, D.J. C. M. P. G. K. B. P. M. P. 1995; Full Text PDF PubMed Scopus Google have recently been that a receptor the to and responsive elements. for the receptors and as to separated by 3 the and receptors have been shown to to direct repeats with between (including and as as to binding to these motifs is to the C. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the receptor to a in to the the VDR, PXR, and CAR to the and form heterodimers with responsive motifs of a be into motifs, including direct everted and Several have that CAR and PXR CYP2 or genes the same responsive elements in a for PXR is to genes of the phenobarbital-responsive DR4 element J.L. S. B. 2000; PubMed Scopus Google and CAR is to human the PXR-responsive elements and B. Moore L.B. Stoltz McKee D.D. Kliewer S.A. Mol. Pharmacol. Google Scholar). The of a between these two nuclear receptor has been This in the ability of a nuclear receptor to but is to from the of the and/or the or the The that VDR binds to and transactivates DR4 and ER6 motifs in to the more DR3 elements are not and of this nuclear receptor Indeed, motifs have been previously including DR4 VDR a for and the A. P. C. Biochem. J. 2000; PubMed Scopus Google Scholar, J. 1999; PubMed Google Scholar). In with of the nuclear receptor that VDR and PXR the in (4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar). The of these nuclear receptors in in contrast to in Indeed, VDR not activated by or by and PXR CAR activated by this is with the that the in the of VDR and PXR is the the of induction of CYP3A4, CYP2B6, and expression in response to with the binding to and transactivation of the and elements by VDR 1 and This the that or of a in the nuclear receptor half-site region is to both the and the between the both the binding of the receptor and ability to with the transcription and/or the various or have shown that the expression of PXR, and CAR is the control of the receptor in primary human J.-M. Drocourt L. J. Maurel P. Vilarem M. Mol. Pharmacol. 2000; PubMed Scopus Google Scholar, J.-M. S. J.M. Maurel P. Vilarem M.-J. Mol. Pharmacol. 2000; PubMed Scopus Google Scholar, J.-M. Drocourt L. S. J.M. Maurel P. Vilarem M.-J. J. Biochem. PubMed Scopus Google Scholar). VDR expression is by this receptor as is not a fully activated receptor is a for induction by observed in the same that the expression of PXR and CAR J.-M. M. J.M. Maurel P. Vilarem M.-J. Biochem. Biophys. Res. Commun. 2000; 274: PubMed Scopus Google to a in CYP2 and gene The present and P. J.M. Mol. Pharmacol. PubMed Scopus Google Scholar, C. K. J. T. Y. H. E. J. E. Mol. Pharmacol. PubMed Scopus Google Scholar) that vitamin D gene expression the of those to with the of in the of the basal expression of CYP2 and genes at in through VDR In the presence of xenobiotics to PXR or these receptors compete with VDR CYP gene responsive elements. In this has to be that the of CYP3A4 and CYP2B6 induction in primary human in response to in response to the that the of VDR basal expression is at of vitamin CYP2B6 to be the of vitamin D these genes is In this is that to be a primary gene S. M. J.-M. L. Vilarem M.-J. Maurel P. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google the expression of normal is at a and this for the that and vitamin induction of this gene is D be from A. E. J. 1999; 277: PubMed Google Scholar). including and of vitamin vitamin are with these is the in induces the of to by to vitamin It is that in and/or for in basal expression and as and as as and for the of the these genes are by VDR is CYP2B6, and CYP3A4 have not been shown to be in the of vitamin D M. PubMed Scopus Google Scholar, 13: PubMed Scopus Google Scholar, K. H. Y. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). has been observed that with vitamin D G. I. Clin. Pharmacol. PubMed Scopus Google Scholar). In of D and by and In and phenobarbital are two of the with a metabolic by a of to an of vitamin D E. A. 1998; Google Scholar). 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Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T-cell leukemia (ATL). One way to address the pathology of the disease lies on conducting research with a molecular approach. In addition to the analysis of ATL-relevant signaling pathways, understanding the regulation of important and relevant transcription factors allows researchers to reach this fundamental objective. HTLV-1 encodes for two oncoproteins, Tax and HTLV-1 basic leucine-zipper factor, which play significant roles in the cellular transformation and the activation of the host's immune responses. Activating protein-1 (AP-1) transcription factor has been linked to cancer and neoplastic transformation ever since the first representative members of the Jun and Fos gene family were cloned and shown to be cellular homologs of viral oncogenes. AP-1 is a dimeric transcription factor composed of proteins belonging to the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra1, and Fra2), and activating transcription factor protein families. Activation of AP-1 transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. AP-1 is also involved in various cellular events including differentiation, proliferation, survival, and apoptosis. Deregulated expression of AP-1 transcription factors is implicated in various lymphomas such as classical Hodgkin lymphomas, anaplastic large cell lymphomas, diffuse large B-cell lymphomas, and adult T-cell leukemia. Here, we review the current thinking behind deregulation of the AP-1 pathway and its contribution to HTLV-induced cellular transformation.

Mammalian cells express sensors able to detect specific features of pathogens and induce the interferon response, which is one of the first line of defenses against viruses and help controlling viral replication. The mechanisms and impact of SARS-CoV-2 sensing in lung epithelial cells remained to be deciphered. In this study, we report that despite a high production of type I and III interferons specifically induced by MDA-5-mediated sensing of SARS-CoV-2, primary and immortalized lung epithelial cells are unable to control viral replication. However, exogenous interferons potently inhibited replication, if provided early upon viral exposure. A better understanding of the ambiguous interplay between the interferon response and SARS-CoV-2 replication is essential to guide future therapeutical interventions.

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

The recent observation made in our laboratory that cellular myc (c-myc) mRNA has a very short half-life in a variety of normal and transformed human cells emphasized the potential importance of post-transcriptional regulation of c-myc gene expression. Jonak and Knight [Jonak, G. J. & Knight, E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 1747-1750] have reported a selective reduction of c-myc mRNA accumulation in lymphoblastoid Daudi cells treated with human beta interferon. This provided a suitable situation in which to examine a possible action of negative modulators of c-myc expression at the level of mRNA stability. Our results confirm the observation by Jonak and Knight that c-myc mRNA level is depressed in cells treated with beta interferon and extend it to alpha 2 interferon. Furthermore, we now demonstrate that interferon has no effect on c-myc transcription rate in isolated nuclei but rather reduces the half-life of its mRNA. Conversely, we show that it increases the level of HLA-A2 mRNA by stimulating its transcription.

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

Abstract Aquatic animal diseases are one of the major limiting factors in aquaculture development, with disease emergence forecast to increase with global change. However, in order to treat increasing diseases in a context of global emergence of antimicrobial resistance and strengthening regulations on antimicrobial use, sustainable alternatives are urgently needed. The use of plant supplements to increase fish immunity and disease resistance has gained much popularity within the last decades. The use of functional supplements, such as plants, can also improve growth and feed assimilation, contributing to a better optimization of aquaculture resources (e.g. fish meal inclusion). We conducted a systematic review and meta‐analysis in order to identify the research gaps in the use of plant‐enriched diets in fish aquaculture and estimate, for the first time, the overall efficacy of plant‐enriched diets on fish growth, immunity and disease resistance as well as the effect of intrinsic parameters (fish trophic level, type of plant material, dosage, treatment duration and pathogen species) on the treatment efficacy. We found that plant‐enriched diets significantly enhanced growth, immunity and disease survival of treated fish, regardless of the fish trophic level, treatment duration and type of material used. We also show that plant supplements are a versatile alternative that can benefit different aquaculture sectors (from small‐scale fish farmers to intensive productions). Finally, we observed that studies need to improve the information reported about the plant material used (e.g. origin, identification, chemical composition), in order to allow the comparison of different experiments and improve their repeatability.

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii . We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures.

Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. Here we employ quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in Drosophila embryos. We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We further reveal that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. We propose that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.

BACKGROUND: The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island. CONCLUSIONS: Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of 'supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.

UNLABELLED: Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4(+) T lymphocytes, a mechanism likely contributing to the loss of CD4(+) T cells. In contrast, in productively infected CD4(+) T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4(+) T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread. IMPORTANCE: Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4(+) T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

Mycobacterium abscessus is a rapidly-growing species causing a diverse panel of clinical manifestations, ranging from cutaneous infections to severe respiratory disease. Its unique cell wall, contributing largely to drug resistance and to pathogenicity, comprises a vast panoply of complex lipids, among which the glycopeptidolipids (GPL) have been the focus of intense research. These lipids fulfill various important functions, from sliding motility or biofilm formation to interaction with host cells and intramacrophage trafficking. Being highly immunogenic, the induction of a strong humoral response is likely to select for rough low- GPL producers. These, in contrast to the smooth high-GPL producers, display aggregative properties, which strongly impacts upon intracellular survival. A propensity to grow as extracellular cords allows these low-GPL producing bacilli to escape the innate immune defenses. Transitioning from high-GPL to low-GPL producers involves mutations within genes involved in biosynthesis or transport of GPL. This leads to induction of an intense pro-inflammatory response and robust and lethal infections in animal models, explaining the presence of rough isolates in patients with decreased pulmonary functions. Herein, we will discuss how, thanks to the generation of defined GPL mutants and the development of appropriate cellular and animal models to study pathogenesis, GPL contribute to M. abscessus biology and physiopathology.

Significance The coevolution of mycobacteria and amoebae seems to have contributed to shaping the virulence of nontuberculous mycobacteria in macrophages. We identified a pool of genes essential for the intracellular survival of Mycobacterium abscessus inside amoebae and macrophages and discovered a hot spot of transposon insertions within the orthologous ESX-4 T7SS locus. We generated a mutant with the deletion of a structural key ESX component, EccB 4 . We demonstrate rupture of the phagosomal membrane only in the presence of an intact eccB 4 gene. These results suggest an unanticipated role of ESX-4 T7SS in governing the intracellular behavior of a mycobacterium. Because M. abscessus lacks ESX-1, it is tempting to speculate that ESX-4 operates as a surrogate for ESX-1 in M. tuberculosis .