Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut.

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117(+) T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.

Decoupled post-glacial history in mutualistic plant-insect interactions: insights from the yellow loosestrife (Lysimachia vulgaris) and its associated oil-collecting bees (Macropis europaea and M-fulvipes)

AimWe take a comparative phylogeographical approach to assess whether three species involved in a specialized oil-rewarding pollination system (i.e. Lysimachia vulgaris and two oil-collecting bees within the genus Macropis) show congruent phylogeographical trajectories during post-glacial colonization processes. Our working hypothesis is that within specialized mutualistic interactions, where each species relies on the co-occurrence of the other for survival and/or reproduction, partners are expected to show congruent evolutionary trajectories, because they are likely to have followed parallel migration routes and to have shared glacial refugia. LocationWestern Palaearctic. MethodsOur analysis relies on the extensive sampling of 104 Western Palaearctic populations (totalling 434, 159 and 74 specimens of Lysimachiavulgaris, Macropiseuropaea and Macropisfulvipes, respectively), genotyped with amplified fragment length polymorphism. Based on this, we evaluated the regional genetic diversity (Shannon diversity and allele rarity index) and genetic structure (assessed using structure, population networks, isolation-by-distance and spatial autocorrelation metrics) of each species. Finally, we compared the general phylogeographical patterns obtained. ResultsContrary to our expectations, the analyses revealed phylogeographical signals suggesting that the investigated organisms demonstrate independent post-glacial trajectories as well as distinct contemporaneous demographic parameters, despite their mutualistic interaction. Main conclusionsThe mutualistic partners investigated here are likely to be experiencing distinct and independent evolutionary dynamics because of their contrasting life-history traits (e.g. dispersal abilities), as well as distinct hubs and migration routes. Such conditions would prevent and/or erase any signature of co-structuring of lineages in space and time. As a result, the lack of phylogeographical congruence driven by differences in life-history traits might have arisen irrespective of the three species having shared similar Pleistocene glacial refugia.

The Peroxisomal Enzyme L-PBE Is Required to Prevent the Dietary Toxicity of Medium-Chain Fatty Acids.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells.

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.

Postmortem biochemistry performed on vitreous humor after postmortem CT-angiography.

Postmortem angiography is becoming increasingly essential in forensic pathology as an adjunct to conventional autopsy. Despite the numerous advantages of this technique, some questions have been raised regarding the influence of the contrast agent injected on the results of toxicological and biochemical analyses. The aim of this study was to investigate the effect of the injection of the contrast agent Angiofil®, mixed with paraffin oil, on the results of postmortem biochemical investigations performed on vitreous humor. Postmortem biochemical investigations were performed on vitreous samples collected from bodies that had undergone postmortem angiography (n=50) and from a control group (n=50). Two vitreous samples were analyzed for each group and the results compared. Glucose, urea, creatinine, 3-β-hydroxybutyrate, sodium and chloride were tested. Different values were observed between the first and second samples in each group. However, these differences were not clinically relevant, suggesting that the injection of this contrast agent mixture does not modify the concentration of the analyzed substances in the vitreous humor.

Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death.

Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis.

LINGO1 and LINGO2 variants are associated with essential tremor and Parkinson disease.

Genetic variation in the leucine-rich repeat and Ig domain containing 1 gene (LINGO1) was recently associated with an increased risk of developing essential tremor (ET) and Parkinson disease (PD). Herein, we performed a comprehensive study of LINGO1 and its paralog LINGO2 in ET and PD by sequencing both genes in patients (ET, n=95; PD, n=96) and by examining haplotype-tagging single-nucleotide polymorphisms (tSNPs) in a multicenter North American series of patients (ET, n=1,247; PD, n= 633) and controls (n=642). The sequencing study identified six novel coding variants in LINGO1 (p.S4C, p.V107M, p.A277T, p.R423R, p.G537A, p.D610D) and three in LINGO2 (p.D135D, p.P217P, p.V565V), however segregation analysis did not support pathogenicity. The association study employed 16 tSNPs at the LINGO1 locus and 21 at the LINGO2 locus. One variant in LINGO1 (rs9652490) displayed evidence of an association with ET (odds ratio (OR) =0.63; P=0.026) and PD (OR=0.54; P=0.016). Additionally, four other tSNPs in LINGO1 and one in LINGO2 were associated with ET and one tSNP in LINGO2 associated with PD (P<0.05). Further analysis identified one tSNP in LINGO1 and two in LINGO2 which influenced age at onset of ET and two tSNPs in LINGO1 which altered age at onset of PD (P<0.05). Our results support a role for LINGO1 and LINGO2 in determining risk for and perhaps age at onset of ET and PD. Further studies are warranted to confirm these findings and to determine the pathogenic mechanisms involved.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory.

Immune protection from intracellular pathogens depends on the generation of terminally differentiated effector and of multipotent memory precursor CD8 T cells, which rapidly regenerate effector and memory cells during recurrent infection. The identification of factors and pathways involved in CD8 T cell differentiation is of obvious importance to improve vaccination strategies. Here, we show that mice lacking T cell factor 1 (Tcf-1), a nuclear effector of the canonical Wingless/Integration 1 (Wnt) signaling pathway, mount normal effector and effector memory CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV). However, Tcf-1-deficient CD8 T cells are selectively impaired in their ability to expand upon secondary challenge and to protect from recurrent virus infection. Tcf-1-deficient mice essentially lack CD8 memory precursor T cells, which is evident already at the peak of the primary response, suggesting that Tcf-1 programs CD8 memory cell fate. The function of Tcf-1 to establish CD8 T cell memory is dependent on the catenin-binding domain in Tcf-1 and requires the Tcf-1 coactivators and Wnt signaling intermediates beta-catenin and gamma-catenin. These findings demonstrate that the canonical Wnt signaling pathway plays an essential role for CD8 central memory T cell differentiation under physiological conditions in vivo. They raise the possibility that modulation of Wnt signaling may be exploited to improve the generation of CD8 memory T cells during vaccination or for therapies designed to promote sustained cytotoxic CD8 T cell responses against tumors.