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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.

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.

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.

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.

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.

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.