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Patterns of Psychotherapy Attendance in Emerging and Mature Adults

Fri, 11/28/2014 - 2:57pm

Approximately 760,000 emerging adults use outpatient psychotherapy in the U.S. each year (Olfson et al.,2002). Emerging adults are 1.6-7.9 times more likely to drop out of mental health treatment than fully mature adults (Edlund et al., 2002; Olfson et al., 2002). This Study compared temporal patterns of attendance and non attendance between emerging and mature adults

Gender: An Important Factor in the Implementation of Services for Juvenile Offenders

Fri, 11/28/2014 - 2:57pm

The Child Welfare League of America (2003) reported that between 1980 and 2000 the arrest rate for boys declined by 11% but increased for girls by 35%. A well tested case management approach being applied more commonly in juvenile justice is the Risk-Needs-Responsivity (RNR) approach, which suggests that interventions and services should be commensurate with ones level of risk and specific dynamic risk factors (criminogenic needs). The RNR model tends to be seen as "gender-neutral", based on assumption that it works equally well with both sexes. Few studies have examined whether gender differences exist in the effectiveness of RNR-type case planning.

Vitopoulos et al., (2012) examined possible RNR differences between justice-involved boys and girls using the Youth Level of Service/Case Management Inventory (YLS/CMI). Across all of the criminogenic need areas (e.g. antisocial attitudes, peer affiliations), only the personality domain was significantly different by gender, such that more girls than boys seemed to have a problem inthis area. They did not find any gender differences in the matching of services to needs identified; however, a higher match between clinician-recommended needs and assigned treatment services (service-to-needs match) predicted a decrease in boys' re-offending but not in girls' reoffending. Given the paucity of research, we are left to question the applicability of some RNR principles or the quality of their implementation for girl offenders. Using the Structured Assessment of Violence Risk for Youth (SAVRY)) in three probation officies to measure both risk level and dynamic risk factors (criminogenic needs), we examined whether within a large sample of youth there were gender differences in the (a) criminogenic needs identified, (b) ability of probation officers (POs) to match services to needs in their case planning and (c) the association of the serve-need match to recidivism.

Relationships as the Foundation of Shared Decision Making: The Experience of Young Adults with Mental Health Conditions

Fri, 11/28/2014 - 2:57pm

To describe the experience of young adults with serious mental illness as active participants in making medication decisions with their psychiatrists.

Relationships as Key to Recovery for Perinatal Women Living with Depression

Fri, 11/28/2014 - 2:57pm

Findings from a study of women with lived experience of depression during and after pregnancy, specific to what is helpful, what are barriers and how to affect change.

Using Life Coaches to Provide Vocational Supports to Emerging Adults

Fri, 11/28/2014 - 2:57pm

Findings presented on using life coaches to provide vocational supports to emerging adults including statistics, types of services, a curriculum and skills inventory.

Genetic and pharmacological reactivation of the mammalian inactive X chromosome

Fri, 11/21/2014 - 1:34pm

X-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. However, trans-acting factors that mediate XCI remain largely unknown. Here, we perform a large-scale RNA interference screen to identify trans-acting XCI factors (XCIFs) that comprise regulators of cell signaling and transcription, including the DNA methyltransferase, DNMT1. The expression pattern of the XCIFs explains the selective onset of XCI following differentiation. The XCIFs function, at least in part, by promoting expression and/or localization of Xist to the Xi. Surprisingly, we find that DNMT1, which is generally a transcriptional repressor, is an activator of Xist transcription. Small-molecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, which has implications for treatment of Rett syndrome and other dominant X-linked diseases. A homozygous mouse knockout of one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect but surprisingly is phenotypically normal. Remarkably, X-linked genes are not overexpressed in female Stc1(-/-) mice, revealing the existence of a mechanism(s) that can compensate for a persistent XCI deficiency to regulate X-linked gene expression.

MicroRNA-378 controls classical brown fat expansion to counteract obesity

Fri, 11/21/2014 - 1:34pm

Both classical brown adipocytes and brown-like beige adipocytes are considered as promising therapeutic targets for obesity; however, their development, relative importance and functional coordination are not well understood. Here we show that a modest expression of miR-378/378* in adipose tissue specifically increases classical brown fat (BAT) mass, but not white fat (WAT) mass. Remarkably, BAT expansion, rather than miR-378 per se, suppresses formation of beige adipocytes in subcutaneous WAT. Despite this negative feedback, the expanded BAT depot is sufficient to prevent both genetic and high-fat diet-induced obesity. At the molecular level, we find that miR-378 targets phosphodiesterase Pde1b in BAT but not in WAT. Indeed, miR-378 and Pde1b inversely regulate brown adipogenesis in vitro in the absence of phosphodiesterase inhibitor isobutylmethylxanthine. Our work identifies miR-378 as a key regulatory component underlying classical BAT-specific expansion and obesity resistance, and adds novel insights into the physiological crosstalk between BAT and WAT.

A therapeutically targetable mechanism of BCR-ABL-independent imatinib resistance in chronic myeloid leukemia

Fri, 11/21/2014 - 1:34pm

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL(+) cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCeta, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration-approved MEK inhibitor, synergistically kills BCR-ABL(+) IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells.

KRAS(G12D)- and BRAF(V600E)-induced transformation of murine pancreatic epithelial cells requires MEK/ERK-stimulated IGF1R signaling

Fri, 11/21/2014 - 1:34pm

Mutation of KRAS is a common initiating event in pancreatic ductal adenocarcinoma (PDAC). Yet, the specific roles of KRAS-stimulated signaling pathways in the transformation of pancreatic ductal epithelial cells (PDEC), putative cells of origin for PDAC, remain unclear. Here, we show that KRAS(G12D) and BRAF(V600E) enhance PDEC proliferation and increase survival after exposure to apoptotic stimuli in a manner dependent on MEK/ERK and PI3K/AKT signaling. Interestingly, we find that activation of PI3K/AKT signaling occurs downstream of MAP-ERK kinase (MEK), and is dependent on the autocrine activation of the insulin-like growth factor (IGF) receptor (IGF1R) by IGF2. Importantly, IGF1R inhibition impairs KRAS(G12D)- and BRAF(V600E)-induced survival, whereas ectopic IGF2 expression rescues KRAS(G12D)- and BRAF(V600E)-mediated survival downstream of MEK inhibition. Moreover, we show that KRAS(G12D)- and BRAF(V600E)-induced tumor formation in an orthotopic model requires IGF1R. Interestingly, we show that while individual inhibition of MEK or IGF1R does not sensitize PDAC cells to apoptosis, their concomitant inhibition reduces survival. Our findings identify a novel mechanism of PI3K/AKT activation downstream of activated KRAS, illustrate the importance of MEK/ERK, PI3K/AKT, and IGF1R signaling in pancreatic tumor initiation, and suggest potential therapeutic strategies for this malignancy.

CRISPRseek: A Bioconductor Package to Identify Target-Specific Guide RNAs for CRISPR-Cas9 Genome-Editing Systems

Fri, 11/21/2014 - 1:34pm

CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3.0 at http://www.bioconductor.org.

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene

Fri, 11/21/2014 - 1:34pm

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG Island Methylator phenotype

Fri, 11/21/2014 - 1:34pm

Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

UMCCTS Newsletter, November 2014

Wed, 11/19/2014 - 11:06am

This is the November 2014 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.

Phosphorylation of rhodopsin by protein kinase C in vitro

Wed, 11/19/2014 - 9:18am

Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.

Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography

Wed, 11/19/2014 - 9:18am

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.

Change in state of nerve growth factor receptor. Modulation of receptor affinity by wheat germ agglutinin

Wed, 11/19/2014 - 9:18am

The binding of 125I-labeled nerve growth factor (NGF) to human melanoma cell (A875) membranes, detergent-soluble membrane extracts, and membrane extracts reconstituted into phospholipid vesicles was significantly increased when binding was carried out in the presence of wheat germ agglutinin (WGA). In the absence of WGA, all 125I-NGF binding was rapidly eliminated by trypsin treatment or rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, in the presence of WGA, up to 75% of 125I-NGF bound was resistant to trypsin digestion and was only slowly dissociated by a high concentration of unlabeled NGF. The effects of WGA can be blocked or reversed by N-acetylglucosamine. Both WGA and NGF rapidly associate with soluble extracts and reconstituted vesicles and, at the concentrations used here, reach binding equilibrium within 2 min. The conversion to slowly dissociating, trypsin-resistant binding, however, was not complete for at least 10 min. Both WGA and NGF are required for maximum accumulation of trypsin-resistant, slowly dissociating binding. The order of addition of NGF and WGA has no effect on the rate of conversion of NGF-receptor, and the conversion occurs after both NGF and WGA are present. The amount of conversion is dependent on the incubation temperature, and significantly greater conversion occurs at 37 than at 0 degrees C. The generation of the trypsin-resistant, slowly dissociating state of NGF-receptor is consistent with a time- and temperature-dependent conformational change in NGF-receptor which occurs after interaction of both NGF and WGA with the receptor or closely associated structures.

Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N

Wed, 11/19/2014 - 9:18am

beta-Adrenergic receptors from turkey erythrocyte membranes have been purified 1000-4000-fold using alprenolol-Sepharose affinity chromatography. Addition of deoxycholate solubilized egg phosphatidylcholine to the beta-adrenergic receptor, that is 5-10% pure and in 0.1% digitonin, followed by Sephadex G-50 gel filtration in buffers containing 30 mM MgCl2 results in 65-70% of the receptor being incorporated into phospholipid vesicles. The beta-adrenergic receptor as detected by photoaffinity labeling using [125I]azidobenzylpindolol in membranes and after alprenolol-Sepharose chromatography is a Mr = 40,000 peptide. Addition of deoxycholate extracts of human erythrocyte membranes, which contain the guanine nucleotide stimulatory regulatory protein of adenylate cyclase (Ns) but not beta-adrenergic receptor, were used to reconstitute a guanine nucleotide-mediated change in agonist affinity for the receptor. These results demonstrate that the alprenolol-Sepharose affinity purified beta-adrenergic receptor is functional in both ligand binding and coupling to Ns. The procedure is rapid, efficient and should be generally applicable to beta-adrenergic receptor and Ns from several different membrane systems.

Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the beta-adrenergic receptor in turkey erythrocytes

Wed, 11/19/2014 - 9:18am

Incubation of turkey erythrocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) results in a dose- and time-dependent desensitization of isoproterenol-stimulated adenylate cyclase activity. Compared to controls, membranes from PMA-treated cells have an isoproterenol-stimulated adenylate cyclase activity that is decreased 20%-40%, with little effect on forskolin or fluoride activation of adenylate cyclase. No change in beta-adrenergic receptor number is observed after PMA treatment, indicating that the major effect of PMA is to uncouple receptor interactions with Ns, the stimulatory guanine nucleotide regulatory protein of adenylate cyclase. Purification of beta-adrenergic receptors from 32Pi-labeled turkey erythrocytes, incubated in the presence or absence of PMA, indicates that the phorbol ester is capable of inducing a 3-fold increase in phosphorylation of the beta-adrenergic receptor. The PMA effect is similar to the phosphorylation of the beta-adrenergic receptor during isoproterenol- and dibutyryl cAMP-induced desensitization of adenylate cyclase in turkey erythrocytes. The findings indicate that decreased receptor-Ns coupling is correlated with receptor phosphorylation and that phorbol esters can influence the responsiveness of hormone-sensitive adenylate cyclase in certain cell types.

Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C

Wed, 11/19/2014 - 9:18am

Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.

Ability of guanine nucleotide derivatives to bind and activate bovine transducin

Wed, 11/19/2014 - 9:18am

Several guanine nucleotide analogs, in one series of which a hydrogen on the 2-amino group is replaced with the p-n-butylphenyl group (BuPGNP derivatives), were used to probe the GTP binding domain of bovine transducin. The order of apparent binding affinities in a series of nucleoside 5'-triphosphates was GTP gamma S greater than GTP approximately BuPGTP greater than dGTP approximately ITP much greater than ATP, values which were 30-100 times higher than affinities of the corresponding 5'-diphosphates. A derivative bearing a 6-aminohexylamino group on the gamma-phosphate, BuPGTP X C6, had a 60-fold lower affinity compared to BuPGTP. In contrast, the p-n-butylphenyl substituent on the 2-amino group had little effect on the binding affinity relative to GTP. Substitutions at the 2-amino group had little effect on either the hydrolysis of the derivatives by the GTPase activity associated with the alpha-subunit of transducin or the activation of cGMP phosphodesterase. The results indicate that the GTP binding domain of transducin is similar in tertiary structure to the corresponding domain of EF-Tu. The 5'-phosphates of GTP are oriented in the binding site of transducin so that the bulky C6 group of BuPGTP X C6 dramatically interferes with binding. The 2-amino group on the guanine ring is probably located at the periphery of the binding site, with the p-n-butylphenyl substituent of BuPGTP facing outward and only weakly interacting with the protein. BuPGTP should be an excellent parent compound for development of novel probes of G-protein interactions with other cellular proteins involved in receptor signal transduction.