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Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium) as well as an improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations. This work was supported by Fondation pour la Recherche Médicale (DEQ20150734352 to JC), Centre National pour la Recherche Scientifique (CNRS, B.F., A.G., V.K., P.P., P.V., J.C.), Institut National pour la Santé et la Recherche Médicale (INSERM; B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.), Sorbonne Université, Faculté des Sciences et Ingénierie (B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.) Labex Biopsy Investissements d’Avenir, ANR-11-IDEX-0004-02 (B.F., A.G., V.K., P.P., P.V., J.-A.G., J.C.), the Spanish Ministerio de Economía y Competitividad-MINECO (#SAF2017-84060-R-AEI/FEDER-UE; E.M.-G., L.D.-R., R.M.), the Spanish Instituto de Salud Carlos III, RETICS-RTA (#RD12/0028/0023; E.M.-G., L.D.-R., R.M.), the Generalitat de Catalunya, AGAUR (#2017-SGR-669; E.M.-G., L.D.-R., R.M.), ICREA-Acadèmia (#2015) and the Spanish Ministerio de Sanidad, Servicios Sociales e Igualdad, Plan Nacional Sobre Drogas (#PNSD-2017I068) to R.M., Fundació La Marató-TV3 (#2016/20-30) to E.M.-G. Fondation pour la Recherche Médicale (FRM#DPA20140629798) and ANR (ANR-16-CE16-0018) to J.A.G.

Introduction Medical therapy in TakoTsubo Syndrome (TTS) remains mainly empirical, given the lack of randomized studies evaluating different pharmacological strategies. The prognostic benefit of angiotensin-converting-enzyme inhibitors and angiotensin II receptor blocker (ACEI/ARB) is not well established. The clinical data published so far are often based on small sample registries and offer opposite results, both in terms of survival and TTS recurrence. Expert recommendations seem favorable to the prescription of ACEI and ARB. Nevertheless, clinical investigation is encouraged for validating the observed results. Purpose The aim of our study was to evaluate the long-term prognostic impact of renin-angiotesin blockers (ACEI or ARB) in terms of mortality and TTS recurrence. Methods The data analyzed in this study were obtained from the nationwide registry “RETAKO”. It included TTS post-discharge survivors, between January 1, 2003, and July 31, 2018. A total of 1062 patients were included for analysis. Cox regression analysis and inverse probability weighting (IPW) propensity score analysis were performed to asses the prognostic benefit of ACEI/ARB. Primary endpoint was a composite outcome of all-cause mortality and TTS recurrence. Results A total of 1062 TTS patients were included. ACEI or ARB were used in 639 patients (60.2%). During a mean follow-up of 2.7±3.5 years, there were 101 deaths (3.9 per 100 patients/year) and 34 recurrences of TTS (1.3 per 100 patients/year). We found no significant difference in follow-up mortality or TTS recurrence in unadjusted and adjusted Cox regression analysis (Hazard Ratio [HR] 0.69, 95% Confidence Interval [CI] 0.47–1.02) between patients treated and untreated with ACEI/ARB. After performing propensity score matching, differences in long term prognosis (all-cause mortality or recurrence) remained no statistically significant (HR 0.73, 95% CI 0.45–1.18). Conclusions In this observational study, we found that ACEI and ARB therapy was not significantly associated with improved long term survival free of recurrence in post-discharged TTS patients. Funding Acknowledgement Type of funding sources: None. Incidence of primary endpoint

Background Chemokine ligand-20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of CC-chemokine receptor 6 (CCR6)-expressing subsets of DCs, B-cells and memory T-cells into the skin. CCL20 and CCR6+ immune cells have been detected in chronic inflammatory skin diseases and several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for melanoma progression remains controversial. Objective The functional contribution of CCR6-expressing immune cell subsets and local CCL20 in the tumor microenvironment for the immune control of melanoma was studied. Methods Homeostatic and inducible CCL20 secretion of murine (B16, Ret) and human (A375, C32) melanoma cells was analyzed by ELISA. To assess the functional relevance of CCR6/CCL20 interactions on local tumor progression, prestimulated or retrovirally transduced B16/F1 melanoma cells overexpressing CCL20 (B16-CCL20) were injected subcutaneously into C57BL/6 Wt mice and congenic CCR6-deficient (CCR6−/−) mice. Infiltrating leucocytes were examined by flow cytometry in tumors and draining lymph nodes (DLNs). Results Melanoma cell lines up-regulate CCL20 secretion upon stimulation with pro-inflammatory cytokines in vitro. While only moderate changes in phenotype and composition of leucocytes were detected in advanced tumors and DLNs, mice injected with CCR6+ B16-CCL20 cells developed smaller tumors compared to B16-Control injected littermates, with CCR6-/- mice displaying the most pronounced reduction in tumor growth and incidence. Conclusion Our results suggest that CCR6/CCL20 interactions and individual independent effects of CCL20 and CCR6 in the microenvironment may be essential for melanoma progression and suggest a decisive role of this chemokine axis for melanoma pathogenesis beyond chemoattraction.

The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota–gut–brain axis) has led to a paradigm shift in the neurosciences. Understanding the neurobiological mechanisms supporting the relevance of actions mediated by the gut microbiota for brain physiology and neuronal functioning is a key research area. In this review, we discuss the literature showing how the microbiota is emerging as a key regulator of the brain’s function and behavior, as increasing amounts of evidence on the importance of the bidirectional communication between the intestinal bacteria and the brain have accumulated. Based on recent discoveries, we suggest that the interaction between diet and the gut microbiota, which might ultimately affect the brain, represents an unprecedented stimulus for conducting new research that links food and mood. We also review the limited work in the clinical arena to date, and we propose novel approaches for deciphering the gut microbiota–brain axis and, eventually, for manipulating this relationship to boost mental wellness.

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express respectively the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially up-regulated in the NAc. We then focused on miR-1 considering the important role of some of its predicted mRNA targets, such as fosb and npas4, in the effects of cocaine. We validated these targets in vitro and in vivo and further showed that overexpression of miR-1 in D1-SPNs of the DS reduced cocaine-induced CPP reinstatement, whereas it increased cue-induced reinstatement of cocaine-SA, without affecting other cocaine-mediated adaptive behavior. In addition, miR-1 overexpression in D2-SPNs of the DS reduced the motivation to self-administer cocaine but did not modify other measured behaviors. Together, our results highlight a precise cell-type- and region-specific control of relapse to cocaine-seeking behaviors by miR-1, and illustrate the importance of cell-specific investigations.

The myxoma virus has become of interest in human medicine in the last two decades as it has the ability to infect many types of human cancer cells and is being used as a platform to develop viro-therapeutic agents that suppress aggressive and damaging immune responses and inflammation. Furthermore, the myxoma virus encodes proteins that have strong immunosuppressive effects, and several of the myxoma virus-encoded immunomodulators are being developed to treat systemic inflammatory syndromes such as cardiovascular disease and transplant rejection. Myxoma virus encodes the M-T7 protein, the most abundantly secreted protein expressed in myxoma virus-infected cells, originally identified as a rabbit species-specific interferon-gamma (IFN-γ) receptor homolog and as a chemokine-modulating protein binding a wide range of mammalian chemokines. M-T7 is a critical virulence factor for viral pathogenesis that increases virus lethality when expressed. Although M-T7 has been extensively studied using biochemical and biophysical techniques and its interactome map is well known, its three-dimensional (3D) structure remains elusive. Obtaining the 3D structure of M-T7 would be greatly beneficial and is a crucial step toward advancing M-T7 research through understanding the molecular function and activity of M-T7 as a novel therapeutic reagent and to rationally develop this protein as a drug. This chapter provides an overview of the structural determination techniques, especially X-ray crystallography, that can be applied toward the goal of achieving the first high-resolution structure of M-T7. In addition, details of up-and-coming methods are discussed, including X-ray diffraction at X-ray free electron lasers (XFELs), nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM), Micro-electron diffraction (Micro-ED), and small-angle X-ray scattering (SAXS), and their potential applications to M-T7 structural biology.

Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds., Trypanosoma brucei inosine-5′-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 Å room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

Interactions of proline-rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein–protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high-affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation–π and salt-bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family.

Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma associated with chemoresistance and a poor prognosis. Various nonsynonymous mutations in the R172 residue of IDH2 are present in 20% to 30% of AITL patients. In addition to their diagnostic value, these mutations are potentially targetable, especially by isocitrate dehydrogenase (IDH) 2 inhibitor, and therefore their identification in a routine setting is clinically relevant. However, in AITL, the neoplastic cells may be scarce, making the identification of molecular anomalies difficult. We evaluated the diagnostic value of different methods to detect IDH2 mutations in formalin-fixed, paraffin-embedded tumor samples. Immunohistochemistry with an anti-IDH2 R172K antibody, Sanger sequencing, high-resolution melting PCR, allele-specific real-time quantitative PCR, and next-generation sequencing (NGS) were applied to biopsy specimens from 42 AITL patients. We demonstrate that the IDH2 R172K antibody is specific to this amino acid substitution and highly sensitive for the detection of the IDH2R172K variant, the most frequent substitution in this disease. In our study, NGS and allele-specific real-time quantitative PCR displayed a good sensitivity, detecting 96% and 92% of IDH2 mutations, respectively, in contrast to Sanger sequencing and high-resolution melting PCR, which showed a significantly lower detection rate (58% and 42%, respectively). These results suggest that a combination of immunohistochemistry and AS-PCR or NGS should be considered for the identification of IDH2 mutations in AITL in a routine setting.

Background Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. Conclusions MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data. Electronic supplementary material The online version of this article (10.1186/s12915-018-0524-5) contains supplementary material, which is available to authorized users.

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.

Integrative research including facies characterization, ichnological composition and foraminifer analysis has been conducted on cores from Site U1385 of the IODP Expedition 339 to evaluate the incidence of Marine Isotope Stage (MIS) 12 and MIS 11 on deep-sea environmental changes. Four color facies groups have been differentiated, showing variable transitions between them (bioturbated, gradual and sharp contacts). Trace fossil assemblage, assigned to the Zoophycos ichnofacies, consists of light and dark filled structures, with Alcyonidiopsis, Chondrites, Nereites, Planolites, Spirophyton, Thalassinoides, Thalassinoides-like structures, and Zoophycos. A deep-sea multi-tiered trace fossil community is interpreted, revealing predominance of well-oxygenated bottom and pore-waters, as well as abundance of food in the sediment for macrobenthic tracemaker community. Changes in environmental parameters are interpreted to be associated with significant variations in trace fossil distribution according to the differentiated intervals (A to M). Benthic foraminifer concentration in the sediments and variations of the planktonic foraminifer assemblages suggest significant changes in surface productivity and food supply to the sea floor since the ending of MIS 13 to the end of MIS 11 that could be correlated with the registered changes in facies and trace fossil assemblages. At the end of MIS 13 values of annual export productivity were very low, that together with the presence of light-color sediments and the continuous presence of light Planolites and Thalassinoides, reveal lower organic carbon flux to the bottom and high oxygen conditions (interval A). Afterwards the organic matter supply increased rapidly and remained very high until Termination V, determining an eutrophic environment, expressed by high benthic foraminifer accumulation rates, and reduced availability of oxygen, that correlate with the record of Spirophyton and Zoophycos, and the presence of Chondrites, observed in intervals B and D. Lower benthic foraminifer accumulation rates during MIS 11 suggest an oligotrophic environment at the bottom consistent with lower inputs of organic carbon, associated with high oxygen content of bottom waters that agrees with the lighter color of the sediments as well as by the continuous presence of light Planolites and Thalassinoides in the differentiated interval M. The evolution of the macrobenthic tracemaker community during MIS 12 and MIS 11 responds to major changes in bottom water ventilation probably linked to variations in deep water (North Atlantic) thermohaline circulation, determining variations in oxygen and food availability.

Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6–7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels. The isolated holoenzyme exhibited the subunit patterns of F-type ATP synthases containing a 5-subunit hydrophilic F1 subcomplex and a 3-subunit hydrophobic FO subcomplex. ATP hydrolysis by the isolated HF1FO ATP synthase was successfully detected after pretreatment with different detergents by an in-gel ATPase activity assay, which showed that the highest activity was detected in the presence of mild detergents such as LDAO; moreover, high catalytic activity in the gel was already detected after the initial incubation period of 0.5 h. In contrast, HF1FO showed extremely low ATPase activity in harsher detergents such as TODC. The isolated fully functional enzyme will form the basis for future structural studies.

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains of NSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1) in vitro. To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1. We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD(+)-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren-Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a "trade-off" system to balance the key role of farnesal as a JH precursor with its potential toxicity.

Biophysical journal 112(3), 506a - 507a (2017). doi:10.1016/j.bpj.2016.11.2740, Published by Cell Press, Cambridge, Mass.

Dothistroma needle blight (DNB) caused by Dothistroma septosporum and Dothistroma pini is a damaging disease of pine in many countries. The disease led to the abandonment of planting susceptible Pinus species in parts of Africa, Asia, Australasia, Europe and North America. Although the disease can be effectively controlled using copper fungicides, this chemical is only routinely applied in forests in New Zealand and Australia. Other management tactics aimed at making conditions less favourable for disease development, such as thinning or pruning, may be effective on some, but not all, sites. Disease avoidance, by planting non-susceptible species, is the most common form of management in Europe, along with deployment of hosts with strong disease resistance. Although D. septosporum is present almost everywhere Pinus is grown, it is important that an effort is maintained to exclude introductions of new haplotypes that could increase virulence or enable host resistance to be overcome. A global strategy to exclude new introductions of Dothistroma and other damaging forest pathogens, facilitated by collaborative programmes and legislation, is needed.

B-phycoerythrin from the red alga Porphyridium cruentum was crystallized using the technique of capillary counter-diffusion. Crystals belonging to the space group R3 with almost identical unit cell constants and diffracting to 1.85 and 1.70 Å were obtained at pH values of 5 and 8, respectively. The most important difference between structures is the presence of the residue His88α in two different conformations at pH 8. This residue is placed next to the chromophore phycoerythrobilin PEB82α and the new conformation results in the relocation of the hydrogen-bond network and hydration around PEB82α, which probably contributes to the observed pH dependence of the optical spectrum associated with this chromophore. Comparison with the structures of B-phycoerythrin from other red algae shows differences in the conformation of the A-ring of the chromophore PEB139α. This conformational difference in B-phycoerythrin from P. cruentum enables the formation of several hydrogen bonds that connect PEB139α with the chromophore PEB158β at the (αβ)(3) hexamer association interface. The possible influence of these structural differences on the optical spectrum and the ability of the protein to perform energy transfer are discussed, with the two pH-dependent conformations of His88α and PEB82α being proposed as representing critical structural features that are correlated with the pH dependence of the optical spectrum and transient optical states during energy transfer.

The hepatitis C virus nonstructural 5A (NS5A) protein is a large zinc-binding phosphoprotein that plays an important role in viral RNA replication and is involved in altering signal transduction pathways in the host cell. This protein interacts with Fyn tyrosine kinase in vivo and regulates its kinase activity. The 1.5 Å resolution crystal structure of a complex between the SH3 domain of the Fyn tyrosine kinase and the C-terminal proline-rich motif of the NS5A-derived peptide APPIPPPRRKR has been solved. Crystals were obtained in the presence of ZnCl(2) and belonged to the tetragonal space group P4(1)2(1)2. The asymmetric unit is composed of four SH3 domains and two NS5A peptide molecules; only three of the domain molecules contain a bound peptide, while the fourth molecule seems to correspond to a free form of the domain. Additionally, two of the SH3 domains are bound to the same peptide chain and form a ternary complex. The proline-rich motif present in the NS5A protein seems to be important for RNA replication and virus assembly, and the promiscuous interaction of the Fyn SH3 domain with the NS5A C-terminal proline-rich peptide found in this crystallographic structure may be important in the virus infection cycle.

International audience; MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.

Class V myosins are dimeric actin-associated motor proteins that deliver cellular cargoes to discrete cellular locations. Fission yeast possess two class V myosins, Myo51 and Myo52. Although Myo52 has been shown to have roles in vacuole distribution, cytokinesis and cell growth, Myo51 has no as yet discernible function in the vegetative life cycle. Here, we uncover distinct functions for this motor protein during mating and meiosis. Not only does Myo51 transiently localise to a foci at the site of cell fusion upon conjugation, but overexpression of the Myo51 globular tail also leads to disruption of cell fusion. Upon completion of meiotic prophase Myo51 localises to the outside of the spindle pole bodies (SPBs), where it remains until completion of meiosis II. Association of Myo51 with SPBs is not dependent upon actin or the septation initiation network (SIN); however, it is dependent on a stable microtubule cytoskeleton and the presence of the Cdc2-CyclinB complex. We observe a rapid and dynamic exchange of Myo51 at the SPB during meiosis I but not meiosis II. Finally, we show that Myo51 has an important role in regulating spore formation upon completion of meiosis.

International audience; We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. In vitro, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8+ cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors in vivo were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney U tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-κB activation. Interleukin-8 (IL-8) was the main cytokine produced in vitro. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8+ T cells in vitro. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment. [Cancer Res 2008;68(22):9423–32]

Intracellular movement is a fundamental property of all cell types. Many organelles and molecules are actively transported throughout the cytoplasm by molecular motors, such as the dimeric type V myosins. These possess a long neck, which contains an IQ motif, that allow it to make 36-nm steps along the actin polymer. Live cell imaging of the fission yeast type V myosin Myo52 reveals that the protein moves rapidly throughout the cytoplasm. Here, we describe analysis of this movement and have established that Myo52 moves long distances on actin filaments in an ATP-dependent manner at ∼0.5 μm/second. Myo51 and the microtubule cytoskeleton have no discernable role in modulating Myo52 movements, whereas rigour mutations in Myo52 abrogated its movement. We go on to show that, although dimerisation is required for Myo52 movement, deleting its neck has no discernable affect on Myo52 function or velocity in vivo.

The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

p140/KIR3DL2 has been identified in malignant cell lines isolated from the skin and blood of patients with transformed mycosis fungoides (MF) and Sezary's syndrome (SS). For the first time, the expression of a cell membrane structure appeared to be able to distinguish CD4+ tumour lymphocytes from reactive lymphocytes in these small cutaneous T-cell lymphomas (CTCLs). This study has examined the in vivo expression of this receptor in various CTCL subtypes, which constituted a heterogeneous group. Tumour cells diffusely expressed KIR in SS, in lymphomatoid papulosis (LyP) and in CD4+CD30+ as well as CD8+ large cell pleomorphic CTCL. In contrast, the infiltrating lymphocytes did not express KIR in MF at the patch/plaque stage or in CD4+CD30- large cell pleomorphic CTCL, except for scattered small cells. One quarter of the transformed MF tested exhibited KIR+ tumour cells, suggesting heterogeneity in this subtype. KIR expression was also examined in inflammatory lesions characterized by a dense infiltrate of T cells, such as lupus erythematosus and lichen planus. Only scattered CD8+ cells in lichen planus expressed a significant amount of KIR3DL2. Taken together, these results show for the first time that KIR molecules are expressed in distinct subtypes of malignant CTCL. It is also shown for the first time that SS and MF, which are frequent variants of CTCL with similar histological features, can be distinguished by their KIR3DL2 expression analysis. The identification of this KIR also differentiates between lupus erythematosus and lichen planus, which are both diseases with dense benign lymphocytic infiltrates.

Background: In 5 to 10% of adult patients with asthma, aspirin (acetylsalicylic acid [ASA]) and most other nonsteroidal anti-inflammatory drugs (NSAIDs) precipitate acute asthmatic attacks. Therefore, choosing an alternative anti-inflammatory agent for patients with adverse reactions to an NSAID is a common problem in clinical practice. The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX that is involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins have provided a reasonable basis for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents. Objective: The purpose of this study was to demonstrate that rofecoxib, a specific inhibitor of COX-2, does not cause asthmatic attacks in patients with ASA and/or other NSAID-induced asthma. Methods: We studied 40 patients, all of whom had experienced asthma induced by at least two different NSAIDs. The patients were challenged in a single-blind manner with different doses of rofecoxib on 3 different days, until either the therapeutic dose of 25 mg or intolerance was reached. Each patient was rechallenged with 25 mg of rofecoxib 7 days later if no evidence of intolerance had been observed previously. Results: Rofecoxib, 25 mg, was proven to be well tolerated in all 40 patients with ASA-induced and NSAID-induced asthma. Conclusion: Our study appears to demonstrate that rofecoxib is a suitable NSAID for treatment of patients with ASA and/or other NSAID-induced asthma. (CHEST 2002; 121:1812–1817)

Hepatosplenic gamma delta T-cell lymphoma (HS gamma delta TCL) is a rare and aggressive subtype of peripheral T-cell lymphoma that has been associated cytogenetically with the isochromosome 7q [i(7)(q10)]. The incidence of this aberration and its relevance to pathogenesis of HS gamma delta TCL is still unknown. We investigated the status of chromosome 7 in 12 HSTCL cases, including nine with a typical gamma delta phenotype, one with a so-called T-cell receptor (TCR)-silent phenotype, and two with the variant alpha beta phenotype. We analyzed available fresh and archival material using a dual-color interphase fluorescence in situ hybridization (FISH) approach with 7p and 7q probes. A significant population of cells with predominance of 7q signals was detected in 10 cases (eight gamma delta, one alpha beta, and one TCR silent), and two lymphomas did not show clonal 7p/7q signal imbalances. In four of 10 cases with chromosome 7 aberrations, a hybridization pattern indicative of the presence of one chromosome 7 and one i(7)(q10) was found. In four other cases, the configuration of signals (2 x 7p/3 x 7q) suggested the presence of the i(7)(q10) and additional structural aberrations involving the second chromosome 7. In two cases, including one alpha beta phenotypic variant, a variety of FISH patterns equivalent to two to five copies of i(7)(q10) or numerical and structural aberrations of second chromosome 7 has been detected. These findings support cytogenetic data pointing to a characteristic association of i(7)(q10) with HSTCL, irrespective of the immunophenotype of malignant cells. An increased number of 7q signals was found in three cases with cytologic features of progression, indicating a tendency of HSTCL to multiply the i(7)(q10) chromosome during evolution.

The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3-9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50-60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.

The product of the Lymphocyte Activation Gene-3 (LAG-3, CD223) is a high affinity MHC class II ligand expressed by activated CD4(+) and CD8(+) T cells, which can associate with the T cell receptor (TCR) and downregulate TCR signalling in vitro. We have also reported that a soluble mLAG-3Ig fusion protein works as a vaccine adjuvant in vivo in mice, enhancing Th1 and CD8 T cell responses. Here, we report that LAG-3 expression was found, using fluorescent activated cell sorting (FACS) analysis, on 11-48% of human tumour-infiltrating lymphocytes (TILs) isolated from eight freshly dissociated renal cell carcinomas (RCCs), and was restricted mostly to CD8(+) cells. Immunohistochemical analysis confirmed LAG-3 expression by TILs in 9/11 RCCs, as well as in tumours of different origins, such as melanomas (3/5) and lymphomas (7/7). Since not only antigen presenting cells (APCs), but also TILs themselves strongly express major histocompatibility complex (MHC) class II, we firstly investigated whether LAG-3/MHC class II T-T cell contacts might influence tumour cell recognition. However, cytotoxicity inhibition was not observed in two RCC-specific CD8(+) T cell clones in the presence of the LAG-3-specific MAb, and there was also no observed difference in the recognition of LAG-3-transfected or wild-type RCC by these cytotoxic T lymphocytes (CTLs). In contrast, MHC class II engagement by LAG-3Ig was found to enhance the capacity of immature dendritic cells to stimulate naive T cell proliferation and IL-12-dependent IFN-gamma production by T cells in vitro. These results therefore provide support for a role for TIL-expressed LAG-3 in the engagement of class II molecules on APCs, thereby contributing to APC activation and Th1/Tc1 commitment, without downregulating cytotoxicity.

Hepatosplenic gammadelta T-cell lymphoma is a recently identified entity in which lymphoma cells bearing the gammadelta T-cell receptor (TCR) infiltrate the sinusoids of the liver and the sinuses of the splenic red pulp and bone marrow, without lymph node involvement. It is also characterized by a recurrent cytogenetic finding, isochromosome 7q (i7q10). The authors report a case of hepatosplenic lymphoma of alphabeta T-cell phenotype that shares the same clinical, histologic, and cytogenetic characteristics of the previously described hepatosplenic gammadelta T-cell lymphoma. Fluorescent in situ hybridization performed with chromosome 7 probes showed the typical pattern of isochromosome 7q. Genomic analysis of the TCR gamma locus failed to detect a clonal rearrangement. This unique case of hepatosplenic lymphoma of alphabeta T-cell phenotype supports the possibility that lymphoid populations of different alphabeta or gammadelta phenotype that share similar homing and presumably functional properties could give rise to lymphomas displaying similar clinical and pathologic findings.

In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

International audience; Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most frequent T-cell lymphoma entities. Follicular helper T lymphocytes (TFH) are recognized as the normal cellular counterpart of the neoplastic component. Despite a clonal T-cell feature and few described recurrent cytogenetic abnormalities, a driving oncogenic event has not been identified so far. It has been recently reported that in mice, heterozygous inactivation of Roquin/Rc3h1, a RING type E3 ubiquitine ligase, recapitulates many of the clinical, histological, and cellular features associated with human AITL. In this study we explored whether ROQUIN alterations could be an initial event in the human AITL oncogenic process. Using microarray and RT-PCR analyses, we investigated the levels of ROQUIN transcripts in TFH tumor cells purified from AITL (n = 8) and reactive tonsils (n = 12) and found similar levels of ROQUIN expression in both. Moreover, we also demonstrated that ROQUIN protein was expressed by AITL TFH (PD1+) cells. We then analysed ROQUIN coding sequence in 12 tumor cell-rich AITL samples and found no mutation in any of the samples. Finally, we analysed the expression of MiR101, a putative partner of ROQUIN involved in the modulation of ICOS expression and found similar levels of expression in tumor and reactive TFH. Altogether, this study shows that neither alteration of ROQUIN gene nor deregulation of miR101 expression is likely to be a frequent recurrent event in AITL.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell–associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

11 pags, 4 figs. -- Supporting Information is available at the Publisher's web. -- Correction: Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR. PLOS ONE 9(4): e95818. https://doi.org/10.1371/journal.pone.0095818, PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution., This work was supported by the Spanish Ministry of Economic Affairs and Competitiveness (www.mineco.gob.es) grants CTQ2011-28680 to FJB and BIO2009-13265-CO2-01 to IL, and by the grant BIPEDD-CM (P-BIO-0214-2006) from Comunidad Autonoma de Madrid (www.madrid.org) to RCO. ADB was supported by a Juan de la Cierva contract from the Spanish Ministry of Economic Affairs and Competitiveness. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; The L-phenylalanine oxidase IL4I1 inhibits T-cell proliferation in vitro through H(2) O(2) production, and is highly expressed in tumor-associated macrophages. IL4I1 is also detected by immunohistochemistry in neoplastic cells from several B-cell lymphomas and some non-lymphoid tumors. To evaluate IL4I1's effect on tumor growth, we developed a mouse melanoma model constitutively coexpressing IL4I1 and the GP33 epitope. After GP33 vaccination, tumors developed more frequently in mice injected with IL4I1-expressing cells in comparison with mice receiving control cells. Tumor escape was preceded by a rapid diminution of IFN-γ-producing cytotoxic antitumor CD8(+) T cells. Moreover, tumor incidence was already increased when only 20% of the injected cells expressed IL4I1. The minimal IL4I1 activities leading to tumor escape were close to those detected in human melanoma and mesothelioma. Thus, we demonstrate the immunosuppressive functions of IL4I1 in vivo and suggest that IL4I1 facilitates human tumor growth by inhibiting the CD8(+) antitumor T-cell response.

The cause of the Rubinstein-Taybi syndrome (RTS), a multiple congenital anomalies/mental retardation (MCA/MR) syndrome first described in 1963, remains obscure. Recently, a deletion of chromosomal material at 16p13.3 has been found in some patients with the disorder, but no such deletion can be identified in the majority of affected individuals. Although the disorder has been well documented to be concordant in at least 7 monozygotic twin pairs and in one non-twin sib pair, only one clear-cut case of parent-to-child transmission has been reported previously. We present here a mother and daughter, both of whom appear to be affected with RTS, strongly suggesting either autosomal or X-linked dominant transmission. The paucity of previous cases of parent-to-child transmission may be related to either decreased fertility or decreased fitness in affected individuals.

Biopsies and cell lines of natural killer/T-cell lymphoma, nasal type (NKTCL) were subject to combined gene expression profiling and array-based comparative genomic hybridization analyses. Compared with peripheral T-cell lymphoma, not otherwise specified, NKTCL had greater transcript levels for NK-cell and cytotoxic molecules, especially granzyme H. Compared with normal NKcells, tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, Epstein-Barr Virus–induced genes, and PDGFRA. Notably, platelet-derived growth factor receptor α and its phosphorylated form were confirmed at the protein level, and in vitro the MEC04 NKTCL cell line was sensitive to imatinib. Deregulation of the AKT, Janus kinase–signal transducers and activators of transcription, and nuclear factor-κB pathways was corroborated by nuclear expression of phosphorylated AKT, signal transducers and activators of transcription 3, and RelA in NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 [1q44], IL6R [1q21.3], CCL2 [17q12], TNFRSF21 [6p12.3]). Several features of NKTCL uncovered by this analysis suggest perturbation of angiogenic pathways. Integrative analysis also evidenced deregulation of the tumor suppressor HACE1 in the frequently deleted 6q21 region. This study highlights emerging oncogenic pathways in NKTCL and identifies novel diagnostic and therapeutic targets.

International audience; Rare cases of peripheral T-cell lymphomas with follicular growth pattern (PTCL-F) have been recently reported, and their association with t(5;9)(q33;q22) involving ITK and SYK has been suggested. However, the clinicopathologic aspects of PTCL-F are poorly described and the normal cell counterpart of this subgroup of lymphoma is still unknown. Therefore, we analyzed the pathologic, phenotypic, and cytogenetic features of a series of 30 patients (range: 33 to 88 y) that showed histopathologic features of PTCL-F in at least 1 biopsy (n=30), either at initial presentation (n=26) or at relapse (n=4). Neoplastic cells were medium-sized clear cells that were CD4+ (24/27, 89%), CD10+ (21/29, 72%), BCL-6+ (14/19, 74%), and expressed programed death-1 (27/27, 100%), CXCL13 (23/27, 85%), and ICOS (11/11, 100%), markers of follicular helper T cells (TFH). Four of 22 patients (18%) had t(5;9)(q33;q22) detected by fluorescence in situ hybridization. Patients with clinical data available had multiple lymphadenopathies (25/28, 89%), stage III to IV diseases (17/26, 65%), B symptoms (7/27, 26%), and skin lesions (6/23, 26%). Three patients with sequential biopsies disclosed clinical and histopathologic features of angioimmunoblastic T-cell lymphoma at initial presentation. Our results show that this rare form of PTCL-F (1) has an immunophenotype indicative of derivation from TFH cells, (2) is associated with t(5;9) in a proportion of cases, and (3) shows some overlapping features with angioimmunoblastic T-cell lymphoma, raising the question of a possible relationship.

The inhibitors of growth (ING) family of tumor suppressors consists of five homologous proteins involved in chromatin remodeling. They form part of different acetylation and deacetylation complexes and are thought to direct them to specific regions of the chromatin, through the recognition of H3K4me3 (trimethylated K4 in the histone 3 tail) by their conserved plant homeodomain (PHD). We have determined the crystal structure of ING4-PHD bound to H3K4me3, which reveals a tight complex stabilized by numerous interactions. NMR shows that there is a reduction in the backbone mobility on the regions of the PHD that participate in the peptide binding, and binding affinities differ depending on histone tail lengths Thermodynamic analysis reveals that the discrimination in favor of methylated lysine is entropy-driven, contrary to what has been described for chromodomains. The molecular basis of H3K4me3 recognition by ING4 differs from that of ING2, which is consistent with their different affinities for methylated histone tails. These differences suggest a distinct role in transcriptional regulation for these two ING family members because of the antagonistic effect of the complexes that they recruit onto chromatin. Our results illustrate the versatility of PHD fingers as readers of the histone code.

Oval cells participate in liver regeneration when hepatocyte replication is impaired. These precursor cells proliferate in periportal regions and organize in ductules. They are surrounded by a basement membrane, the degradation of which by matrix metalloproteinases (MMP) might trigger their terminal differentiation into hepatocytes. We studied the expression of MMP-2 and MMP-9 and that of one of their tissue inhibitors (TIMP-1) in a model of hepatic regeneration from precursor cells. Regeneration was induced by treating rats with 2-acetylaminofluorene followed by partial hepatectomy. MMP-2 and MMP-9 hepatic expression paralleled oval cell number with a peak at day 9-14 after hepatectomy. They were mainly detected in oval cells. TIMP-1 mRNA and oncostatin M receptor mRNA, a major regulator of TIMP-1 synthesis, markedly increased from day 1 after surgery until day 9 and then declined; they were mainly detected in interlobular bile duct cells and oval cells until day 14. In agreement with the in vivo data, the WB-F344 liver precursor cell line expressed MMP-2 and MMP-9, as well as TIMP-1 and oncostatin M receptor. These data suggest that (a) early increased TIMP-1 synthesis by biliary and oval cells favors basement membrane deposition around proliferating ductular structures through MMP inhibition, (b) delayed increased MMP expression, concomitant to decreased TIMP-1 synthesis, leads to basement membrane degradation, preceding oval cell differentiation, (c) the oncostatin M pathway might play a major role in increased TIMP-1 synthesis.

Background & Aims: The growth arrest-specific gene 6 (Gas6) protein is a vitamin K-dependent protein that binds to the Axl subfamily of tyrosine kinase receptors and exerts antiapoptotic and proliferative effects. Because Gas6 plays a role in development and tissue remodelling, we studied its expression as well as that of its high-affinity receptor Axl in a well-characterized model of hepatic regeneration from precursor oval cells. Methods: Hepatic regeneration was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. Results: Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 and 14 after hepatectomy and declined thereafter. Oval cells expressed Gas6 protein and messenger RNA (mRNA). Axl mRNA hepatic levels paralleled the number of oval cells, and immunohistochemistry showed Axl expression in these cells. WB-F344 cells, a hepatocytic precursor cell line, also expressed Gas6 and Axl. Addition of Gas6 significantly increased the number of WB-F344 cells cultured with or without serum. Gas6 did not increase cell entry in the S phase of the cell cycle but inhibited 15-d-prostaglandin J 2 -induced WB-F344 cell apoptosis. Conclusions: Our data demonstrate an expression of Gas6 and of its receptor Axl by oval cells during hepatic regeneration. Because the Gas6/Axl couple protects from apoptosis a hepatocytic precursor cell line, these results strongly suggest that the Gas6/Axl couple favors oval cell accumulation in regenerating liver by an autocrine/paracrine mechanism.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive MCA-MR disorder caused by mutations within the 7-dehydrocholesterol reductase gene, DHCR7. The diagnosis is based on the biochemical findings of elevated plasma 7-dehydrocholesterol (7DHC) levels. It is a panethnic condition with variable mutation frequencies in different populations. Ten Cuban patients and four Canadian patients of Mediterranean ancestry with SLOS are reported herein. All these patients are at the mild end of the clinical spectrum (the highest Kelley-Hennekam severity score was 28 in one patient). All patients had genotypes which were compound heterozygous or homozygous for T93M; in all the Mediterranean patients the T93M mutation appeared to be associated with the J haplotype. Another compound heterozygote for T93M was of Ukrainian/Irish ancestry; in this patient the T93M was associated with a new haplotype designated K. The T93M mutation was initially reported as the most common in a series of patients from Italy. Our identification of a single haplotype associated with the T93M mutation in patients whose ancestors originate in the region of the Mediterranean Sea basin suggests a founder effect.

Novel DNA minor-groove binding ligands with a promising antibacterial profile are described. Apart from excellent in vitro potency against multiple Gram-positive bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and penicillin-intermediate Streptococcus pneumoniae (PISP), a small subset of compounds was active against Gram-negative bacteria such as Escherichia coli (E. coli).