query: RTEL1 interacts with TRF2 through a C4C4 motif

title: TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

text: The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase.

title: The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins

text: Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.

title: De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation.

text: Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.

title: Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5

text: The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 Å resolution. This α/β protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13° bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three α-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel β-structure and random coil that interacts with the minor groove.

title: Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats.

text: Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.

title: PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening.

text: The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.

title: PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication

text: Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication. Here, we report that replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1–Cul4DDB1 ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator.

title: Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting.

text: We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1-3 in the HEK293 cell mRNA 3' UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3' UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

title: Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

text: Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-α/β and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3′ non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5′ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP–RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.

title: CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms.

text: CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.

title: Two sequence motifs from HIF-1alpha bind to the DNA-binding site of p53.

text: There is evidence that hypoxia-inducible factor-1alpha (HIF-1alpha) interacts with the tumor suppressor p53. To characterize the putative interaction, we mapped the binding of the core domain of p53 (p53c) to an array of immobilized HIF-1alpha-derived peptides and found two peptide-sequence motifs that bound to p53c with micromolar affinity in solution. One sequence was adjacent to and the other coincided with the two proline residues of the oxygen-dependent degradation domain (P402 and P564) that act as switches for the oxygen-dependent regulation of HIF-1alpha. The binding affinity was independent of the hydroxylation state of P564. We found from NMR spectroscopy that these sequence motifs bind to the DNA-binding site of p53c. Because the two sequences are homologous and separated by 120 residues, and one is in a largely unstructured transactivation domain, we speculate that each sequence motif in HIF-1alpha binds to a different subunit of the p53 tetramer, leading to very tight binding. The binding data support the proposal that p53 provides a route for the degradation in hypoxic tumor cells of HIF-1alpha that is not hydroxylated at the two proline residues.

title: A coactivator of pre-mRNA splicing.

text: The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.

title: Functional elements within the dynein microtubule-binding domain

text: Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379, 80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated.

title: ESCRT-III recognition by VPS4 ATPases

text: The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1–3 class of ESCRT-III proteins. Structures of VPS4A MIT–CHMP1A and VPS4B MIT–CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.

title: The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage

text: It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.

title: Telomeric 3′ Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST

text: A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.

title: A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C.

text: Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

title: A vertebrate gene, ticrr, is an essential checkpoint and replication regulator.

text: Eukaryotes have numerous checkpoint pathways to protect genome fidelity during normal cell division and in response to DNA damage. Through a screen for G2/M checkpoint regulators in zebrafish, we identified ticrr (for TopBP1-interacting, checkpoint, and replication regulator), a previously uncharacterized gene that is required to prevent mitotic entry after treatment with ionizing radiation. Ticrr deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. We show that the human TICRR ortholog associates with TopBP1, a known checkpoint protein and a core component of the DNA replication preinitiation complex (pre-IC), and that the TICRR-TopBP1 interaction is stable without chromatin and requires BRCT motifs essential for TopBP1's replication and checkpoint functions. Most importantly, we find that ticrr deficiency disrupts chromatin binding of pre-IC, but not prereplication complex, components. Taken together, our data show that TICRR acts in association with TopBP1 and plays an essential role in pre-IC formation. It remains to be determined whether Ticrr represents the vertebrate ortholog of the yeast pre-IC component Sld3, or a hitherto unknown metazoan replication and checkpoint regulator.

title: A beginning of the end: new insights into the functional organization of telomeres

text: Ever since the first demonstration of their repetitive sequence and unique replication pathway, telomeres have beguiled researchers with how they function in protecting chromosome ends. Of course much has been learned over the years, and we now appreciate that telomeres are comprised of the multimeric protein/DNA shelterin complex and that the formation of t-loops provides protection from DNA damage machinery. Deriving their name from D-loops, t-loops are generated by the insertion of the 3' overhang into telomeric repeats facilitated by the binding of TRF2. Recent studies have uncovered novel forms of chromosome end-structure that may implicate telomere organization in cellular processes beyond its essential role in telomere protection and homeostasis. In particular, we have recently described that t-loops form in a TRF2-dependent manner at interstitial telomere repeat sequences, which we termed interstitial telomere loops (ITLs). These structures are also dependent on association of lamin A/C, a canonical component of the nucleoskeleton that is mutated in myriad human diseases, including human segmental progeroid syndromes. Since ITLs are associated with telomere stability and require functional lamin A/C, our study suggests a mechanistic link between cellular aging (replicative senescence induced by telomere shortening) and organismal aging (modeled by Hutchinson Gilford Progeria Syndrome). Here we speculate on other potential ramifications of ITL formation, from gene expression to genome stability to chromosome structure.

title: A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors.

text: The Ndc80 complex is the key microtubule-binding element of the kinetochore. In contrast to the well-characterized interaction of Ndc80-Nuf2 heads with microtubules, little is known about how the Spc24-25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24-25 in complex with the histone-fold protein Cnn1/CENP-T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80-Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1-related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80-binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N-terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters.


query: RUNX is not expressed in skin tissue.

title: Human epidermal stem cell function is regulated by circadian oscillations.

text: Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFβ and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.

title: Response to self antigen imprints regulatory memory in tissues

text: Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.

title: Inhibition of limb regeneration in the axolotl after treatment of the skin with actinomycin D.

text: In this experiment actinomycin D was used to explore the action of the wound epidermis on underlying tissues during limb regeneration. In axolotl forelimbs the skin was removed from the elbow to the shoulder. Skin from the right limbs was soaked for three hours in actinomycin D (5.0 or 10.0 μg/ml 0.6% NaCl). For controls, skin from left limbs was soaked in 0.6% NaCl for the same period of time. Each piece of skin was orthotopically replanted, and both limbs were amputated through the treated skin, proximal to the elbow. After an initial healing period, the control limbs regenerated normally. Except for a slightly paler color, limbs bearing actinomycin-treated skin were indistinguishable from the controls, both grossly and histologically, during the first week following amputation. While the control limbs formed early blastemas, no grossly visible evidence of regeneration was apparent in the experimental limbs, but histologically some dedifferentiation was occurring. Normally three to four digits were seen in the control regenerates before blastemas appeared on the experimental limbs. By 35–40 days blastemas had appeared on most experimental limbs. These developed very rapidly, and within a short time many of them had attained levels of development close to the controls. Actinomycin D temporarily suppresses formation of the apical epidermal cap and the subsequent aggregation of dedifferentiated cells into a blastema. When the effect wears off, an apical cap forms and the dedifferentiated cells quickly organize into a blastema and begin to differentiate.

title: Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype.

text: Hutchinson–Gilford progeria syndrome (HGPS; OMIM 176670) is an extremely rare but devastating disorder that mimics premature aging.1–3 Affected children appear normal at birth but typically develop failure to thrive in the first two years. Other features include alopecia, micrognathia, loss of subcutaneous fat with prominent veins, abnormal dentition, sclerodermatous skin changes, and osteolysis of the clavicles and distal phalanges. The mean age of death is at age 13 years, most commonly due to atherosclerosis. HGPS is mainly sporadic in occurrence, but a genetic cause has now been implicated following the identification of de novo heterozygous mutations in the LMNA gene in the majority of HGPS patients.4,5 A single family showing autosomal recessive inheritance of homozygous LMNA mutations has also been reported.6  LMNA encodes lamins A and C, components of the nuclear lamina, a meshwork underlying the nuclear envelope that serves as a structural support and is also thought to contribute to chromatin organisation and the regulation of gene expression.7,8 Interestingly, mutations in LMNA have recently been associated with at least eight inherited disorders, known as laminopathies, with differential dystrophic effects on a variety of tissues including muscle, neurones, skin, bone, and adipose tissue (reviewed in Mounkes et al 9). However, the realisation that these disorders share common genetic defects has led to clinical re-evaluation, with emerging evidence of significant phenotypic overlap.10 Hence the laminopathies might reasonably be considered as a spectrum of related diseases.   HGPS has phenotypic similarities to several other laminopathies, in particular the atypical Werner’s syndrome11 and mandibuloacral dysplasia (MAD; OMIM 248370 and 608612).12 These diseases are associated with lipodystrophy,3,13 which is the most prominent feature of another laminopathy, familial partial lipodystrophy of the Dunnigan variety (OMIM 151660).14 MAD has been further classified as two …

title: Reprogramming of human somatic cells to pluripotency with defined factors

text: Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.

title: p16INK4A is a robust in vivo biomarker of cellular aging in human skin.

text: The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.

title: Accumulation of hydroxyethyl starch in human and animal tissues: a systematic review

text: To systematically review clinical and preclinical data on hydroxyethyl starch (HES) tissue storage. MEDLINE (PubMed) was searched and abstracts were screened using defined criteria to identify articles containing original data on HES tissue accumulation. Forty-eight studies were included: 37 human studies with a total of 635 patients and 11 animal studies. The most frequent indication for fluid infusion was surgery accounting for 282 patients (45.9 %). HES localization in skin was shown by 17 studies, in kidney by 12, in liver by 8, and in bone marrow by 5. Additional sites of HES deposition were lymph nodes, spleen, lung, pancreas, intestine, muscle, trophoblast, and placental stroma. Among major organs the highest measured tissue concentration of HES was in the kidney. HES uptake into intracellular vacuoles was observed by 30 min after infusion. Storage was cumulative, increasing in proportion to dose, although in 15 % of patients storage and associated symptoms were demonstrated at the lowest cumulative doses (0.4 g kg−1). Some HES deposits were extremely long-lasting, persisting for 8 years or more in skin and 10 years in kidney. Pruritus associated with HES storage was described in 17 studies and renal dysfunction in ten studies. In one included randomized trial, HES infusion produced osmotic nephrosis-like lesions indicative of HES storage (p = 0.01) and also increased the need for renal replacement therapy (odds ratio, 9.50; 95 % confidence interval, 1.09–82.7; p = 0.02). The tissue distribution of HES was generally similar in animals and humans. Tissue storage of HES is widespread, rapid, cumulative, frequently long-lasting, and potentially harmful.

title: Arginine depletion as a mechanism for the immune privilege of corneal allografts

text: The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-L-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

title: Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival

text: The only proven requirement for ascorbic acid (vitamin C) is in preventing scurvy, presumably because it is a cofactor for hydroxylases required for post-translational modifications that stabilize collagen. We have created mice deficient in the mouse ortholog (solute carrier family 23 member 1 or Slc23a1) of a rat ascorbic-acid transporter, Svct2 (ref. 4). Cultured embryonic fibroblasts from homozygous Slc23a1−/− mice had less than 5% of normal ascorbic-acid uptake. Ascorbic-acid levels were undetectable or markedly reduced in the blood and tissues of Slc23a1−/− mice. Prenatal supplementation of pregnant females did not elevate blood ascorbic acid in Slc23a1−/− fetuses, suggesting Slc23a1 is important in placental ascorbic-acid transport. Slc23a1−/− mice died within a few minutes of birth with respiratory failure and intraparenchymal brain hemorrhage. Lungs showed no postnatal expansion but had normal surfactant protein B levels. Brain hemorrhage was unlikely to be simply a form of scurvy since Slc23a1−/− mice showed no hemorrhage in any other tissues and their skin had normal skin 4-hydroxyproline levels despite low ascorbic-acid content. We conclude that Slc23a1 is required for transport of ascorbic acid into many tissues and across the placenta. Deficiency of the transporter is lethal in newborn mice, thereby revealing a previously unrecognized requirement for ascorbic acid in the perinatal period.

title: Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

text: Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

title: NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations.

text: Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.

title: Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8-positive solid lymphomas: a tissue-based variant of primary effusion lymphoma.

text: Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8, is consistently identified in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Here we report four cases of KSHV-bearing solid lymphomas that occurred in AIDS patients (cases 1 to 3) and in a human immunodeficiency virus (HIV)-seronegative person (case 4). The patients presented extranodal masses in the abdomen (cases 1, 3, and 4) or skin (case 2), and nodal involvement, together with Kaposi's sarcoma (case 3). The gastrointestinal tract was involved in two patients (cases 1 and 3). The patients did not develop a lymphomatous effusion. KSHV was detected in the tumor cells of all cases by immunohistochemistry and by polymerase chain reaction. Epstein-Barr virus was detected in two of the HIV-related cases. All KSHV-positive solid lymphomas exhibited PEL-like cell morphology. To investigate the relationship of these disorders to PEL and to other AIDS-associated diffuse large cell lymphomas, KSHV-positive solid lymphomas were tested for the expression of a set of genes that were previously shown by gene profiling analysis to define PEL tumor cells. The results showed that expression of this set of genes in KSHV-positive lymphomas is similar to that of PEL but distinct from KSHV-negative AIDS-associated diffuse large cell lymphomas. Because pathobiological features of KSHV-positive solid lymphomas closely mimic those of PEL, our results suggest that KSHV-positive solid lymphomas should be considered as a tissue-based variant of classical PEL, irrespective of HIV status.

title: The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens

text: The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.

title: Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes

text: Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

title: Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

text: T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC-activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC-imprinted T cells protect against influenza more effectively than do gut and skin DC-imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC-activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC-activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.

title: [Pharmacokinetics and biotransformation of the antimycotic drug ciclopiroxolamine in animals and man after topical and systemic administration].

text: 1. Following the dermal application of the carbon-14 labelled broad spectrum antimycotic 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Hoe 296, Batrafen) in the form of a 1% aqueous cream to healthy human dorsal skin (penetration time: 6 h; occlusive dressing for 5 h), percutaneous absorption accounted on average for 1.3% of the dose applied. Excretion occurred via the kidney, with biological half-lives of 1.7 h. As can be seen from penetration studies of cadaverous skin, the horny layer contained the highest concentrations, with values of 2300-4500 microgram/cm3. The levels determined in the corium were still above the minimum inhibitory concentrations. These concentrations were already obtained at the first test stage (1.5 h after application) and did not change virtually at all over the longer penetration period. According to studies using histoautoradiography, ciclopirox can penetrate the skin via the epidermis and the hair follicles. When ciclopirox-14C-olamine aqueous cream was spread on the surface of fingernails, the radioactive-labelled compound penetrated right through the nail. The percutaneous absorption in dogs was higher, at 5-15% of the dose, than it was in humans. 2. After vaginal application (1 mg/kg) of ciclopirox-14C-olamine in the form of a 1% aqueous cream to bitches, between 42 and 97% of the dose (depending on the animal) was recovered in the urine and faeces, the remainder having penetrated into the tampon used to close the vagina. 3. Ciclopirox is excreted by dogs and man in the urine, primarily as a glucuronide. In humans another glucuronide with properties similar to those of the original substance was detected. Two conjugated, relatively non-polar metabolites were also present in small amounts. The metabolite patterns after oral and dermal application were similar. The binding of ciclopirox to serum proteins in humans was 96 +/- 2% in a concentration range of 0.01-11.0 microgram/ml. 4. Placental transfer was low in the rats studied. Though there was good absorption by the mother animal, the radioactivity in the foetal tissues was always lower than that of the maternal blood.

title: Dosimetry and risk estimates of radioiodine therapy for large, multinodular goiters.

text:
UNLABELLED In patients with a large, multinodular goiter (> 100 g), radiation absorbed doses in the thyroid, surrounding tissues and remainder of the body were estimated after therapeutic administration of 131I(3.7 MBq or 100 microCi/g of thyroid tissue retained at 24 hr).
 METHODS Thermoluminescent dosimeter (TLD) measurements were performed on 23 patients (12 euthyroid and 1I hyperthyroid; thyroid weight 222 +/- 72 g; 2.1 +/- 0.9 GBq 131I) on the skin over the thyroid, over the submandibular gland and over the parotid gland. Thyroid radioactivity measurements were done daily in 6 euthyroid and 6 hyperthyroid patients (thyroid weight 204 +/- 69 g; 1.9 +/- 0.9 GBq 131I). An iodine biokinetic model and the MIRD methodology were used to estimate absorbed doses in organs. Cancer risks were calculated using ICRP Publication 60.
 RESULTS Cumulated absorbed doses on the skin (TLD measurements) were 4.2 +/- 1.4 Gy (thyroid), 1.2 +/- 0.6 Gy (submandibular) and 0.4 +/- 0.2 Gy (parotid). All these values were significantly correlated with the amount of radioiodine retained in the thyroid at 24 hr (euthyroid versus hyperthyroid not significant). Absorbed doses in the thyroid of 94 +/- 25 Gy for euthyroid and 93 +/- 17 Gy for hyperthyroid patients were calculated (thyroid radioactivity measurements). Extrathyroidal absorbed doses (means of 12 patients) were 0.88 Gy in the urinary bladder, 0.57 Gy in the small intestine, 0.38 Gy in the stomach, and ranged from 0.05 to 0.30 Gy in other organs (euthyroid versus hyperthyroid not significant). A 1.6% life-time risk of development of cancer outside the thyroid gland was calculated. When applied to people of 65 yr and older the estimated risk is approximately 0.5%.
 CONCLUSION These data may help in choosing the treatment regimen for individual patients with a large, multinodular goiter, who have to be treated for hyperthyroidism or compressive problems. In younger patients, surgery may be preferred. However, for elderly patients and patients with cardiopulmonary disease, the advantages of noninvasive radioiodine treatment will outweight the life-time risk of this mode of therapy.

title: Cell and molecular mechanisms of insulin-induced angiogenesis

text: Angiogenesis, the development of new blood vessel from pre-existing vessels, is a key process in the formation of the granulation tissue during wound healing. The appropriate development of new blood vessels, along with their subsequent maturation and differentiation, establishes the foundation for functional wound neovasculature. We performed studies in vivo and used a variety of cellular and molecular approaches in vitro to show that insulin stimulates angiogenesis and to elucidate the signalling mechanisms by which this protein stimulates microvessel development. Mice skin injected with insulin shows longer vessels with more branches, along with increased numbers of associated alpha-smooth muscle actin-expressing cells, suggesting the appropriate differentiation and maturation of the new vessels. We also found that insulin stimulates human microvascular endothelial cell migration and tube formation, and that these effects occur independently of VEGF/VEGFR signalling, but are dependent upon the insulin receptor itself. Downstream signalling pathways involve PI3K, Akt, sterol regulatory element-binding protein 1 (SREBP-1) and Rac1; inhibition of these pathways results in elimination of endothelial cell migration and tube formation and significantly decreases the development of microvessels. Our findings strongly suggest that insulin is a good candidate for the treatment of ischaemic wounds and other conditions in which blood vessel development is impaired.

title: CRITICAL REVIEW Cutaneous Vasculitis Update: Diagnostic Criteria,

text: Vasculitis, inflammation of the vessel wall, can result in mural destruction with hemorrhage, aneurysm formation, and infarction, or intimal-medial hyperplasia and subsequent stenosis leading to tissue ischemia. The skin, in part due to its large vascular bed, exposure to cold temperatures, and frequent presence of stasis, is involved in many distinct as well as un-named vasculitic syndromes that vary from localized and self-limited to generalized and life-threatening with multi-organ disease. To exclude mimics of vasculitis, diagnosis of cutaneous vasculitis requires biopsy confirmation where its acute signs (fibrinoid necrosis), chronic signs (endarteritis obliterans), or past signs (acellular scar of healed arteritis) must be recognized and presence of extravascular findings such as patterned fibrosis or collagenolytic granulomas noted. Although vasculitis can be classified by etiology, many cases have no identifiable cause, and a single etiologic agent can elicit several distinct clinicopathologic expressions of vasculitis. Therefore, the classification of cutaneous vasculitis is best approached morphologically by determining vessel size and principal inflammatory response. These histologic patterns roughly correlate with pathogenic mechanisms that, when coupled with direct immunofluorescent examination, anti-neutrophil cytoplasmic antibody (ANCA) status, and findings from work-up for systemic disease, allow for specific diagnosis, and ultimately, more effective therapy. Herein, we review cutaneous vasculitis focusing on diagnostic criteria, classification, epidemiology, etiology, pathogenesis, and evaluation of the cutaneous vasculitis patient.

title: Safety and reliability of Radio Frequency Identification Devices in Magnetic Resonance Imaging and Computed Tomography

text:
BACKGROUND Radio Frequency Identification (RFID) devices are becoming more and more essential for patient safety in hospitals. The purpose of this study was to determine patient safety, data reliability and signal loss wearing on skin RFID devices during magnetic resonance imaging (MRI) and computed tomography (CT) scanning.
 METHODS Sixty RFID tags of the type I-Code SLI, 13.56 MHz, ISO 18000-3.1 were tested: Thirty type 1, an RFID tag with a 76 x 45 mm aluminum-etched antenna and 30 type 2, a tag with a 31 x 14 mm copper-etched antenna. The signal loss, material movement and heat tests were performed in a 1.5 T and a 3 T MR system. For data integrity, the tags were tested additionally during CT scanning. Standardized function tests were performed with all transponders before and after all imaging studies.
 RESULTS There was no memory loss or data alteration in the RFID tags after MRI and CT scanning. Concerning heating (a maximum of 3.6 degrees C) and device movement (below 1 N/kg) no relevant influence was found. Concerning signal loss (artifacts 2 - 4 mm), interpretability of MR images was impaired when superficial structures such as skin, subcutaneous tissues or tendons were assessed.
 CONCLUSIONS Patients wearing RFID wristbands are safe in 1.5 T and 3 T MR scanners using normal operation mode for RF-field. The findings are specific to the RFID tags that underwent testing.

title: Epidemiology of Staphylococcus aureus blood and skin and soft tissue infections in the US military health system, 2005-2010.

text:
CONTEXT Rates of hospital-onset methicillin-resistant Staphylococcus aureus (MRSA) infections are reported as decreasing, but recent rates of community-onset S. aureus infections are less known.
 OBJECTIVES To characterize the overall and annual incidence rates of community-onset and hospital-onset S. aureus bacteremia and skin and soft tissue infections (SSTIs) in a national health care system and to evaluate trends in the incidence rates of S. aureus bacteremia and SSTIs and the proportion due to MRSA.
 DESIGN, SETTING, AND PARTICIPANTS Observational study of all Department of Defense TRICARE beneficiaries from January 2005 through December 2010. Medical record databases were used to identify and classify all annual first-positive S. aureus blood and wound or abscess cultures as methicillin-susceptible S. aureus or MRSA, and as community-onset or hospital-onset infections (isolates collected >3 days after hospital admission).
 MAIN OUTCOME MEASURES Unadjusted incidence rates per 100,000 person-years of observation, the proportion of infections that was due to MRSA, and annual trends for 2005 through 2010 (examined using the Spearman rank correlation test or the Mantel-Haenszel χ2 test for linear trend).
 RESULTS During 56 million person-years (nonactive duty: 47 million person-years; active duty: 9 million person-years), there were 2643 blood and 80,281 wound or abscess annual first-positive S. aureus cultures. Annual incidence rates varied from 3.6 to 6.0 per 100,000 person-years for S. aureus bacteremia and 122.7 to 168.9 per 100,000 person-years for S. aureus SSTIs. The annual incidence rates for community-onset MRSA bacteremia decreased from 1.7 per 100,000 person-years (95% CI, 1.5-2.0 per 100,000 person-years) in 2005 to 1.2 per 100,000 person-years (95% CI, 0.9-1.4 per 100,000 person-years) in 2010 (P = .005 for trend). The annual incidence rates for hospital-onset MRSA bacteremia also decreased from 0.7 per 100,000 person-years (95% CI, 0.6-0.9 per 100,000 person-years) in 2005 to 0.4 per 100,000 person-years (95% CI, 0.3-0.5 per 100,000 person-years) in 2010 (P = .005 for trend). Concurrently, the proportion of community-onset SSTI due to MRSA peaked at 62% in 2006 before decreasing annually to 52% in 2010 (P < .001 for trend).
 CONCLUSION In the Department of Defense population consisting of men and women of all ages from across the United States, the rates of both community-onset and hospital-onset MRSA bacteremia decreased in parallel, while the proportion of community-onset SSTIs due to MRSA has more recently declined.


query: RUNX1 is downregulated or mutated in TLX1 T-ALL.

title: Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL

text: The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

title: Myelodysplasia and leukemia of Fanconi anemia are associated with a specific pattern of genomic abnormalities that includes cryptic RUNX1/AML1 lesions.

text: Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), -7/7q (17.2%), and 11q- (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

title: An Extensive MicroRNA-Mediated Network of RNA-RNA Interactions Regulates Established Oncogenic Pathways in Glioblastoma

text: By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3'UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

title: Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification.

text: CBFbeta is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFbeta levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFbeta is also required at very early stages of natural killer (NK)-cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFbeta insufficient cells, nor can overexpression of Runx1 or CBFbeta overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFbeta, and both signals converge to activate the T-cell developmental program.

title: Membrane association of the CD3ε signaling domain is required for optimal T cell development and function.

text: The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.

title: The long non-coding RNA H19-derived miR-675 modulates human gastric cancer cell proliferation by targeting tumor suppressor RUNX1.

text: The lncRNA H19 has been recently shown to be upregulated and play important roles in gastric cancer tumorigenesis. However, the precise molecular mechanism of H19 and its mature product miR-675 in the carcinogenesis of gastric cancer remains unclear. In this study, we found that miR-675 was positively expressed with H19 and was a pivotal mediator in H19-induced gastric cancer cell growth promotion. Subsequently, the tumor suppressor Runt Domain Transcription Factor1 (RUNX1) was confirmed to be a direct target of miR-675 using a luciferase reporter assay and Western blotting analyses. A series of rescue assays indicated that RUNX1 mediated H19/miR-67-induced gastric cancer cell phenotypic changes. Moreover, the inverse relationship between the expression of RUNX1 and H19/miR-675 was also revealed in gastric cancer tissues and gastric cancer cell lines. Taken together, our study demonstrated that the novel pathway H19/miR-675/RUNX1 regulates gastric cancer development and may serve as a potential target for gastric cancer therapy.

title: Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program.

text: At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.

title: Landscape of genetic lesions in 944 patients with myelodysplastic syndromes

text: High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

title: Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators.

text: Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1(+/-)::Gata2(+/-) compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

title: BRAF mutation predicts for MEK-dependence in non-small cell lung cancer (NSCLC).

text: 5247 Constitutive ERK signaling is common in human cancer and is often the result of activating mutations of BRAF, RAS and upstream receptor tyrosine kinases. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (>90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations for MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive with the exception being a cell line with concurrent NRAS and intermediate activity BRAF mutations. Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50’s ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective and prolonged inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between Akt activity and PD0325901 sensitivity was observed. These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and Akt signaling may be required in NSCLC tumors with high basal AKT activity.

title: GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment.

text: Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.

title: IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice.

text: Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

title: Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

text: Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

title: Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL

text: Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

title: The sphingosine-1-phosphate receptor agonist FTY720 modulates dendritic cell trafficking in vivo.

text: The pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled-sphingosine-1-phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1-5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM-1, CD54/ICAM-1 and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.

title: PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome.

text: Recently, mutations in genes involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor have been identified in a new subclass of congenital disorders of glycosylation (CDGs) with a distinct spectrum of clinical features. To date, mutations have been identified in six genes (PIGA, PIGL, PIGM, PIGN, PIGO, and PIGV) encoding proteins in the GPI-anchor-synthesis pathway in individuals with severe neurological features, including seizures, muscular hypotonia, and intellectual disability. We developed a diagnostic gene panel for targeting all known genes encoding proteins in the GPI-anchor-synthesis pathway to screen individuals matching these features, and we detected three missense mutations in PGAP2, c.46C>T, c.380T>C, and c.479C>T, in two unrelated individuals with hyperphosphatasia with mental retardation syndrome (HPMRS). The mutations cosegregated in the investigated families. PGAP2 is involved in fatty-acid GPI-anchor remodeling, which occurs in the Golgi apparatus and is required for stable association between GPI-anchored proteins and the cell-surface membrane rafts. Transfection of the altered protein constructs, p. Arg16Trp (NP_001243169.1), p. Leu127Ser, and p. Thr160Ile, into PGAP2-null cells showed only partial restoration of GPI-anchored marker proteins, CD55 and CD59, on the cell surface. In this work, we show that an impairment of GPI-anchor remodeling also causes HPMRS and conclude that targeted sequencing of the genes encoding proteins in the GPI-anchor-synthesis pathway is an effective diagnostic approach for this subclass of CDGs.

title: Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes.

text: Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.

title: Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia.

text: Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocellular bone marrow. In 40% of DBA patients, various physical anomalies are also present. Currently two genes are associated with the DBA phenotype--the ribosomal protein (RP) S19 mutated in 25% of DBA patients and RPS24 mutated in approximately 1.4% of DBA patients. Here we report the identification of a mutation in yet another ribosomal protein, RPS17. The mutation affects the translation initiation start codon, changing T to G (c.2T>G), thus eliminating the natural start of RPS17 protein biosynthesis. RNA analysis revealed that the mutated allele was expressed, and the next downstream start codon located at position +158 should give rise to a short peptide of only four amino acids (Met-Ser-Arg-Ile). The mutation arose de novo, since all healthy family members carry the wild-type alleles. The identification of a mutation in the third RP of the small ribosomal subunit in DBA patients further supports the theory that impaired translation may be the main cause of DBA pathogenesis.

title: Identification of functional cooperative mutations of SETD2 in human acute leukemia

text: Acute leukemia characterized by chromosomal rearrangements requires additional molecular disruptions to develop into full-blown malignancy, yet the cooperative mechanisms remain elusive. Using whole-genome sequencing of a pair of monozygotic twins discordant for MLL (also called KMT2A) gene–rearranged leukemia, we identified a transforming MLL-NRIP3 fusion gene and biallelic mutations in SETD2 (encoding a histone H3K36 methyltransferase). Moreover, loss-of-function point mutations in SETD2 were recurrent (6.2%) in 241 patients with acute leukemia and were associated with multiple major chromosomal aberrations. We observed a global loss of H3K36 trimethylation (H3K36me3) in leukemic blasts with mutations in SETD2. In the presence of a genetic lesion, downregulation of SETD2 contributed to both initiation and progression during leukemia development by promoting the self-renewal potential of leukemia stem cells. Therefore, our study provides compelling evidence for SETD2 as a new tumor suppressor. Disruption of the SETD2-H3K36me3 pathway is a distinct epigenetic mechanism for leukemia development.

title: Blood stem cells emerge from aortic endothelium by a novel type of cell transition

text: The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.