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Background/Introduction To guide implantable defibrillator (ICD) use in hypertrophic cardiomyopathy (HCM), European Society of Cardiology (ESC) guidelines recommend using individualised sudden death (SCD) risk scores based on quantitative clinical data. Newly published American guidelines are based on the accumulation of binary risk markers, which include imaging-based novel risk markers (NRMs) that were absent from the development of the ESC's algorithm. These NRMs are ejection fraction Purpose To assess how NRMs may have altered ICD prescription across ESC-based SCD risk status prior to publication of current American guidance. Methods We examined electronic records (2013–2020) of a subset of HCM patients with contemporaneous (within 12 months) CMR and echocardiography data for NRMs, ESC risk status, and ICD prescription. Differences in categorical data were assessed by Fisher's exact test. Results We studied 334 HCM patients (74% male; age: 58±14 years), of whom 83 (25%) were referred for ICD. ESC risk status was considered low, medium (4–6% 5-year SCD risk), or high in 264, 26, and 20 patients, for whom ICDs were recommended in 40 (15%), 20 (77%), and 18 (90%) patients, respectively. In patients with low SCD risk status, rate of ICD recommendation was significantly higher when ≥1 NRMs were present (34/126 – 27% vs. 0 NRMs: 6/138 – 4%; p Conclusion NRMs have disproportionate influence on ICD prescription in low ESC risk HCM patients. However, NRMs are not independent of ESC risk status, suggesting iterative development of the ESC's algorithmic approach will be the most effective way of predicting SCD. Funding Acknowledgement Type of funding sources: None.

Contains fulltext : 248786.pdf (Publisher’s version ) (Open Access) Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Many patients with primary focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but were never validated. We aimed to find proteins involved in the mechanism of action of CPF(s) and/or potential biomarkers for the presence of CPF(s). Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s) or healthy and disease controls. Podocyte proteomes were analyzed by LC–MS. Results were validated using flow cytometry, RT-PCR, and immunofluorescence. Podocyte granularity was examined using flow cytometry, electron microscopy imaging, and BODIPY staining. Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, and correlated with the capacity of plasma to induce podocyte granularity, identified as lipid droplet accumulation. Elevated podocyte perilipin-2 was confirmed at protein and mRNA level and was also detected in glomeruli of FSGS patients whose active disease plasmas induced podocyte perilipin-2 and lipid droplets. Our study demonstrates that presumably, CPF-containing plasmas from FSGS patients induce podocyte lipid droplet accumulation and perilipin-2 expression, identifying perilipin-2 as a potential biomarker. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism, which ultimately may result in novel leads for treatment.

The inv(16) acute myeloid leukemia-associated CBFβ-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFβ-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFβ-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFβ-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N(6)-methyladenosine (m(6)A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m(6)A in 18S rRNA at position A(1832). We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m(6)A in rRNA in stemness, differentiation, development, and diseases.

Cement stabilized rammed earth (CSRE) is one of the developed and affordable housing technologies, adopted in many countries, which has been used in Sri Lanka since 2004 for building single and two-floor houses. Steel slip-form and timber formwork have been used for casting walls. Information collected on wall cracks under these two methods is analyzed to justify the reasons for these cracks. Data collected at the initial stage of construction and its serviceability period of 12 years is considered under this study. Most of the walls have been in satisfactory condition, but few had to be repaired. CSRE wall tends to crack similar to other masonry technology due to faulty construction and environmental changes. Major issues are the cracks due to shrinkage of CSRE material which appeared after one year period of construction. Detailing the construction with the provision of shrinkage cracks is required to mitigate the vertical cracks on CSRE walls.

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N6-methyladenosine (m6A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m6A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m6A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m6A and positively regulates mRNA stability in an m6A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m6A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m6A, m6A interactors and mRNA homeostasis.

Accurate and sensitive detection of protein–protein and protein–RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein–protein and protein–RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.

MBD5 and MBD6 are two members of the methyl-CpG-binding domain (MBD) family of proteins that are poorly characterized. Studies performed thus far have failed to show binding of the MBD5 and MBD6 MBD to methylated DNA. Here, we show that both MBD5 and MBD6 interact with the mammalian PR-DUB Polycomb protein complex in a mutually exclusive manner. Strikingly, the MBD of MBD5 and MBD6 is both necessary and sufficient to mediate this interaction. Chromatin immunoprecipitation analyses reveal that MBD6 and FOXK2/PR-DUB share a subset of genomic target genes, suggesting a functional interaction in vivo. Finally, we show that MBD6, but not MBD5, is recruited to sites of DNA damage in a PR-DUB independent manner. Our study thus implies a shared function for MBD5 and MBD6 through an interaction with PR-DUB, as well as an MBD6-specific recruitment to sites of DNA damage.

It is known that researchers have valued the properties of rammed earth walls in terms of natural comfort, durability, affordability and ecofriendliness. Variety of developments has been introduced continually by them for adoption in building construction. As a result, cement stabilized rammed earth (CSRE) and cement stabilized earth bricks (CSEB) are being used today for constructing load bearing walls of houses in many parts of the world. CSRE has been practiced in Sri Lanka from 2004 and a few housing projects have been successfully completed with minimal cost. CSRE walls in randomly selected housing units in a project were investigated after a period of 13 years and found that these walls are in good condition with the protection of surface coating and little maintenance. CSEB wall junctions, soil erosion near the base of walls especially at the external gable walls are identified as defects of CSRE walls. Defects at CSEB wall junctions have been solved in recent research. Good cooling effect in these houses is highly recognized by the users. High level of supervision is required when constructing CSRE to keep its durability, strength, and especially to gain acceptability from the users.

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

Objective In this investigation, the in vitro sustained fluoride release, weight loss and erosive wear of three conventional glass ionomer cements (Fuji IX, ChemFil Superior, Ketac-Silver), three resin-modified glass ionomer cements (Fuji II LC, Vitremer, Photac-Fil), a polyacid-modified resin composite (Dyract), and a resin composite control material (Z100) were compared. Methods The amounts of fluoride released and weight changes were measured for 12 weeks using a fluoride electrode with TISAB III buffer. After 12 weeks, the specimens were recharged with fluoride using 2 mL of 1.23% APF gel. The recharged specimens were assessed for the amounts of fluoride released and weight changes over another 12 weeks. At the end of the experiment, the specimens were examined with SEM and surface profilometry. Results All materials, with the exception of Z100, showed the highest initial fluoride release rates during the first 2 days, dropping quickly over 2 weeks and becoming largely stabilised after 5 weeks, in an exponential mode. The recharging of the specimens with APF gel caused a large increase in the amounts of fluoride released during the first 2 days only. Analyses for all cements showed strong correlations between mean weight loss and cumulative fluoride release over a 5-week period following the application of the APF gel. SEM and surface profilometry found that roughness increased from the polyacid-modified resin composite to the conventional glass ionomer cements. Conclusions APF gel caused erosive wear of the glass ionomer cements especially, and the wear correlated well with the weight losses. To minimise surface erosion, APF gel should not be used on these cements, especially as the recharging effects are transitory.

International audience; The SGF29 protein binds to tri-methylated lysine-4 of histone H3 (H3K4me3), which is a histone modification associated with active promoters. Human SGF29 is a subunit of the histone acetyltransferase module of the SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-Two-A-containing 2A) co-activator complexes. Previous work revealed that the SAGA complex is recruited to endoplasmic reticulum (ER) stress target genes and required for their induction. Here, we report the involvement of SGF29 in the survival of human cells from ER stress. SGF29 knockdown results in impaired transcription of the ER stress genes GRP78 and CHOP. Besides histone H3K14 acetylation, we find that SGF29 is also required for the maintenance of H3K4me3 at these genes, which is already present prior to ER stress. Reduced levels of H3K4me3 in the absence of SGF29 correlate with a decreased association of ASH2L, which is a core component of the SET1/MLL complexes, to GFP78 and CHOP. In conclusion, our results suggest that the H3K4me3-binding protein SGF29 plays a central and dual role in the ER stress response. Prior to ER stress, the protein coordinates H3K4me3 levels, thereby maintaining a 'poised' chromatin state on ER stress target gene promoters. Following ER stress induction, SGF29 is required for increased H3K14 acetylation on these genes, which then results in full transcriptional activation, thereby promoting cell survival.

The process of eukaryotic transcription initiation involves the assembly of basal transcription factor complexes on the gene promoter. The recruitment of TFIID is an early and important step in this process. Gene promoters contain distinct DNA sequence elements and are marked by the presence of post-translationally modified nucleosomes. The contributions of these individual features for TFIID recruitment remain to be elucidated. Here, we use immobilized reconstituted promoter nucleosomes, conventional biochemistry and quantitative mass spectrometry to investigate the influence of distinct histone modifications and functional DNA-elements on the binding of TFIID. Our data reveal synergistic effects of H3K4me3, H3K14ac and a TATA box sequence on TFIID binding in vitro. Stoichiometry analyses of affinity purified human TFIID identified the presence of a stable dimeric core. Several peripheral TAFs, including those interacting with distinct promoter features, are substoichiometric yet present in substantial amounts. Finally, we find that the TAF3 subunit of TFIID binds to poised promoters in an H3K4me3-dependent manner. Moreover, the PHD-finger of TAF3 is important for rapid induction of target genes. Thus, fine-tuning of TFIID engagement on promoters is driven by synergistic contacts with both DNA-elements and histone modifications, eventually resulting in a high affinity interaction and activation of transcription.

A six-year-old female dachshund was examined because of intermittent lameness in its left pelvic limb and periodic back pain. Myelography, epidurography and computed tomography (CT) revealed a dorsal displacement of the dural sac in the lumbosacral region caused by a soft tissue mass which had the specific density of fat. The mass was removed via a dorsal laminectomy in the lumbosacral area and a histological examination confirmed that it was adipose tissue. The clinical signs resolved after the surgery and a follow-up CT five months later showed no evidence of compression of the dural sac. The diagnosis of epidural lipomatosis in this dog was based on the clinical findings, the results of diagnostic imaging, and the surgical and histological findings, all of which revealed many similarities with epidural lipomatosis in man.

Background Histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, DNA repair, and sperm compaction. Modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, Oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture. Results We show that a histone complement of 47 gene loci encodes 31 histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. A particularly diverse array of 15 male-specific histone variants was uncovered, including a testes-specific H4t, the first metazoan H4 sequence variant reported. Universal histone variants H3.3, CenH3, and H2A.Z are present but O. dioica lacks homologs of macroH2A and H2AX. The genome encodes many H2A and H2B variants and the repertoire of H2A.Z isoforms is expanded through alternative splicing, incrementally regulating the number of acetylatable lysine residues in the functionally important N-terminal "charge patch". Mass spectrometry identified 40 acetylation, methylation and ubiquitylation posttranslational modifications (PTMs) and showed that hallmark PTMs of "active" and "repressive" chromatin were present in O. dioica. No obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome. Conclusions These results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. Substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage.

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Summary Induction of varicocele was attempted by ligation of the left renal vein (LRV) in male dogs (Group I). Before the operation and in the 4-month post-operative period, sperm count, sperm motility, and sperm morphology of Group I (n = 8) dogs were compared to sham operated animals (Group 11, n = 5). Furthermore, haemodynamics as well as testicular and vascular morphology were studied. In Group I, changes in diameter and consistency of the spermatic cord were temporary. Semen quality was reduced significantly during the second month after ligation of the LRV, but improved thereafter. Haemodynamic studies revealed that LRV blood pressure was increased significantly in Group I dogs. An extensive venous collateral network replaced the occluded LRV. Retrograde blood flow in the left testicular vein (LTV) was observed only in the proximal part of the LTV of Group I dogs. In Group II dogs numerous pairs of sufficient valves prevented reflux into the LTV. Histological examination revealed that spermatogenesis was not impaired and that the left pampiniform plexus had not changed. The number of Leydig cells was decreased slightly in Group I dogs. Sufficient valves in the LTV prevented formation of a permanent varicocele.

Sexual differentiation of emission rates (Experiment 1) and physical structure of ultrasonic vocalizations (Experiment 2) were investigated in Mongolian gerbils between 17 and 85 days of age. Animals from different litters were allowed to interact in iso- and heterosexual pairs. Vocalization rates increased in all groups, reaching a plateau at approximately day 56. Female pups had significantly higher rates of all vocalizations than male pairs before the plateau stage. Five stereotypic categories of physical structure were easily distinguished and confirmed by sound spectrographic analysis. Only mid-range frequencies of vocalizations appeared to decline throughout the juveniles' lives: this change may communicate the animal's age. Two male and one female categories were sexually dimorphic and similar in structure to those made during courtship interactions. Rates of male-type vocalizations appear to increase as testicular androgens start rising.

The Ras family of oncogenes is activated in a large fraction of human cancers. Treatment of Ras-driven tumors with inhibitors of protein kinases in the Ras signaling network, such as Raf or MEK is a promising therapeutic strategy. However, toxicity issues and the emergence of tumor cells that are resistant to these drugs underscore the need for a better understanding of the Ras pathway and for the development of novel therapeutic options to improve the survival of cancer patients. Deregulation in lysine methylation signaling has emerged as a common etiologic factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumors. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP Kinase signaling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LAC), we found that abrogating SMYD3 catalytic activity inhibits tumor development in response to oncogenic Ras. We employed protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signaling module. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP Kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signaling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signaling. Citation Format: Pawel K. Mazur, Nicolas Reynoird, Purvesh Khatri, Atul J. Butte, Alex Wilkinson, Benjamin Garcia, Shichong Liu, Michiel Vermeulen, Pascal W.T.C. Jansen, Peter J. Tummino, Ryan G. Kruger, Glenn S. Van Aller, Olena Barbash, Michael Huddleston, Or Gozani, Julien Sage. SMYD3 links methylation of MAP3K2 to Ras-driven tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A23.

With recent advances in both imaging techniques and reperfusion therapies, patients with acute stroke now have a realistic window of opportunity for effective intervention and treatment outcome. It is generally believed that “time is brain,” i.e. the maximum benefit can only be achieved when intervention is initiated within the first 3–6 hours after the onset of symptoms, depending upon the treatment (e.g. intravenous administration of tissue plasminogen activator or endovascular recanalization).

In a cohort of 252 Dutch labrador retrievers born between 1988 and 1992, seven founders for fragmented coronoid process were identified. The 185 labrador retrievers born to this cohort between January 1, 1993 and January 1, 1997, were examined clinically, and radiographs of both elbows taken in four directions at 12 to 18 months of age, or earlier when they had signs of lameness, were evaluated. The diagnosis of fragmented coronoid process was confirmed by arthrotomy. The incidence of the condition in the 185 dogs was 17.3 per cent, and for each dog a genetic risk factor was calculated on the basis of its relatedness to the seven founders. The risk factors ranged from 0.07 to 0.41. The dogs were divided into classes of increasing predicted risk, and the mean risk for each class was then compared with the clinical outcome. There were no significant differences between the predicted risk and the outcome in any of the classes.

We report central pontine myelinolysis in orthotopic liver transplant patients. Sequential MR imaging of these patients with central pontine myelinolysis shows progressive decrease of T2-weighted MR signal in the pons, which may not resolve despite complete neurologic recovery.

BackgroundDNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs). Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD) family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA.Methodology/principal findingsFor the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC). In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs) showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif.Conclusions/significanceThe here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

The development of in vivo brain imaging during the past several decades has offered psychiatrists their first opportunity to observe directly both the structure and the metabolic and neurochemical function in the brains of patients suffering from major mental illnesses. Computerized tomography (CT), techniques for measuring regional cerebral blood flew (rCBF), and positron emission tomography (PET) have all documented, for example, that patients suffering from schizophrenia have a variety of cerebral abnormalities (Johnstone et al 1976; Weinberger et al 1979 a,b; Andreasen 1982; Ingvar et al 1974; Weinberger et al 1986; Gur et al 1985; Gur et al 1987; Buchsbaum et al 1982, 1987; Sedvall et al 1986; Early et al 1987; Wong et al 1986). The most commonly observed abnormalities include: 1) ventricular enlargement, 2) decreased cerebral perfusion in the frontal lobes and difficulty in increasing perfusion in response to cognitive challenge, 3) decreased glucose utilization in the frontal cortex and difficulty in activation, and 4) hypermetabolic activity in subcortical regions such as the basal ganglia.

Our current MRI studies grow out of earlier pilot work. In our first MRI study, we examined a sample of 38 schizophrenics and 45 normal controls using principally a midsagittal cut for our analyses. In these studies, we focused our emphasis on morphometrics, or the study of structural size that had potential functional significance. In this first study, we observed a significant decrease in frontal lobe size, cerebral size, and (somewhat to our surprise) cranial size [1]. We were unable to explain this finding on the basis of differences between patients and normal controls and possible confounding variables such as sex, height, or weight. In that study, however, it did appear that there was a possible sex effect in the schizophrenic patients, with slightly more males showing the frontal, cerebral, and cranial abnormalities. These findings were relatively striking. 39 percent of the schizophrenic patients had frontal lobe size outside the control range, while percentages for cerebral and cranial size outside the control range were 25 and 18 percent, respectively. In this study, it also appeared that there were abnormalities in callosal shape in the schizophrenic patients, with a reversal of the “normal” sexual dimorphism that has been observed by other investigators [2]. In this study the female schizophrenic patients had smaller splenia than did the males [3].

5025 Background: The ability to predict efficacy prior to or early during the course of a therapeutic modality can lead to improved outcome by avoiding ineffectual therapies. The purpose of this study was to test if in-vivo functional MR imaging can, by reflecting microcirculatory response of tumor to cytotoxic therapy, predict failure early during the course of treatment in cervical cancer. Methods: One-hundred and one patients with cervical cancer stages IB2-IV treated with radiation/chemotherapy (RT/CT) underwent Dynamic Contrast Enhanced (DCE) MRI before (1st MRI) and during RT at 2 weeks (2nd MRI) and 4 weeks of RT (3rdMRI). Mean follow up was 4.9 years (1.5–8.0 years). Pelvic tumor control and disease-free survival were correlated with imaging parameters derived from the time/signal-intensity curve of the DCE-MRI of each tumor pixel. These included the signal intensity (SI) of the plateau phase (SIp), the lowest 2.5th and 5.0th percentiles of the entire tumor pixels (SI 2.5 and SI 5.0), the slope of...

5095 Background: Pixel-by-pixel analysis of signal intensity (SI) using dynamic contrast enhanced MRI (DCE- MRI) in cervical cancer has been reported to predict treatment outcome. Low enhancement correlates with treatment failure presumably due to poor blood supply/hypoxia. However, within a heterogeneous tumor, the relative volume and degree of low-enhancement regions that correlate with failure is uncertain. This project analyzed the threshold of low-enhancement pixels within the tumor to optimize the predictive power for treatment outcome. Methods: 101 patients with advanced cervical cancer underwent DCE-MRI early during radiation/chemotherapy (2 weeks after therapy start). The SI of each pixel was calculated from the time-intensity curve of the DCE-MRI at the plateau phase. The SI of all pixels within the tumor was plotted as a pixel SI spectrum. The lowest 2.0th through 40th percentiles of the pixel spectrum were used to define the low-enhancement volume, and each percentile category was correlated w...

Magnetic resonance imaging is frequently used for visualization of soft tissues of the head and neck. It was the purpose of this study to determine whether gold eyelid weights, used for treatment of lagophthalmos in facial palsy, would be deflected at high magnetic strengths during imaging. Ex vivo study of six incrementally increasing weights at two magnetic strengths was performed. There was no deflection of any of the gold eyelid loads at either 0.5-tesla or 1.5-tesla magnetic strengths. We conclude that patients with gold eyelid weights are not at risk of damage from movement of the prostheses during magnetic resonance imaging examination.

A detailed vertical gradient magnetic survey of part of a small intrusion known as the Butterton Dyke has been made with the aid of a microcomputer-based data gathering system. The results of over 16 500 magnetic measurements made by one person in less than a day have given results which reveal a detailed pattern of magnetic anomalies. From these it is possible to trace the course of two dykes for a distance of over 300 m, and identify places where they change direction and show small offsets. The advantage of making vertical gradient rather than total field magnetic measurements include a faster surveying speed and better resolution of near-surface anomalies.

Detailed magnetic surveys have been made to see if the form of magnetic anomalies produced by buried metal pipelines can be used to predict the position of joints between individual sections of pipe. This information can help minimize the amount of excavation required when pipelines have to be examined. Magnetic measurements have been made over pipelines where the position of joints is known, others where their position is unknown, and over a range of types and sizes of pipe with either spigot and socket or welded joints. It is shown that in open country the magnetic anomalies over pipelines consist of a sequence of magnetic highs and lows. This pattern is related to the individual sections of pipe from which the pipelines are constructed, and can be used to determine the position of joints between pipe sections. This method of joint location has been unsuccessful in urban areas due to the presence of other disturbing magnetic anomalies, and the sections of pipe not being uniformly magnetized.

The admittance of a parallel−plate capacitor immersed in a warm collisional electron−ion magnetoplasma is studied both experimentally and theoretically. Particular attention is given to the admittance behavior near the lower−hybrid resonance. It is shown that this resonance can be identified experimentally, and therefore that admittance measurements can be used to measure the ion plasma frequency directly, in magnetoplasmas containing one predominant ion species.

To determine the cause of hyperglycemia appearing after pancreas transplantation in type I diabetic recipients, we performed 65 oral glucose tolerance tests with serum insulin and C-peptide determinations in 32 patients with pancreas grafts functioning two or more months following transplantation. We correlated these results with estimates of graft size obtained by magnetic resonance imaging (MRI) and values of urinary amylase as a measure of pancreatic exocrine function. A total of 33 studies were obtained in 20 patients at times of normal glucose tolerance, and normal ranges for serum insulin and C-peptide levels were established; 32 studies in 17 patients during periods of glucose intolerance revealed values of serum insulin and C-peptide that were within the normal range, though the time to peak values was delayed to 2 hr, characteristic of type II diabetes. Only 3 of 17 patients examined by MRI had significant pancreatic allograft atrophy. These patients also had low urinary amylase excretion, and the only values for serum C-peptide that were below the normal range. The other 14 hyperglycemic patients had normalized pancreas grafts, normal urinary amylase excretion, and normal values for serum insulin and C-peptide. In our experience, then, in 76% of patients with hyperglycemia more than 2 months following pancreas transplantation, the cause was appearance of type II diabetes rather than destruction of the allograft with recurrence of type I diabetes. This observation has important implications for the definition of pancreas allograft failure and for the management of pancreas allograft recipients with hyperglycemia.

Manufacturers of geophysical instruments have kept abreast of the many developments in microelectronics that have taken place in the last decade, and have made important developments in geophysical instrumentation. The instruments they have produced are not only smaller and more robust than their predecessors, but are easier to operate, produce a quicker response, and often perform additional functions.

The following notes are for the most part based upon the actual valuation of a registered friendly society and an attempt is made to describe the various operations involved and the points which arise for consideration, from the time the actuary (or valuer as he is commonly known in friendly society circles) is approached, to the time he signs his report and presents it to the society. I have, however, digressed occasionally from the main subject of these notes in order to express my views on certain general points which frequently arise in the valuations of friendly societies. It should be understood that these notes do not necessarily embody official opinion.The specimen society is a typical “centralised” rural society (i.e. without branches) of about 1,000 members, their occupations being described as domestic, horticultural and agricultural.