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A large literature has formed around the question of how Freud's Jewishness and/or Judaism influenced his psychological discoveries and development of psychoanalytic theory and methods. The article organizes the literature into several core theses but brings new clarity and insight by applying two essential criteria to demonstrate an impact of Judaism on Freud's thinking: direct content and historical timing. First, there should be evidence that Freud incorporated actual content from Jewish sources, and second, this incorporation must have occurred during the most crucial period of Freud's early discovery, conceptualization, and development of psychoanalysis, roughly 1893-1910. Thus, for example, Bakan's well-known theory that Freud studied Kabbala is completely negated by the absence of any evidence in the required time period. Part I reviews the literature on the influence of Freud's ethnic/cultural Jewish identity. Part II introduces the Judaic sacred literature, explores Freud's education in Judaism and Hebrew, and presents evidence that Freud had the motive, means, and resources to discover and draw from the "Dream Segment" of the Talmud-along with the traditional Judaic methods and techniques of textual exegesis. Freud then applied these same Judaic word-centered interpretive methods-used for revealing an invisible God-to revealing an invisible Unconscious in four successive books in 1900, 1901, and 1905., (© 2023 Wiley Periodicals LLC.)

Only 8% of US public schools operate their own community water systems, and thus are subject to the federal Lead and Copper Rule's regulation of water lead levels (WLLs). To date, the absence of parallel water testing data for all other schools has prevented the comparison of WLLs with schools that do not face federal regulation. This study compiled and analyzed newly available school-level WLL data that included water source (on-site well water or public utility) and pipe material data for public schools in New York State located outside of New York City. Despite direct federal regulation, schools that used water from on-site wells had a substantially higher percentage of water fixtures with elevated WLLs. Schools that used both on-site well water and iron pipes in their water distribution system had the highest percentage of elevated fixtures. Variation in water treatment practices was identified as a potential contributing mechanism, as schools that used on-site well water were less likely to implement corrosion control. The study concluded that information about water source and premise plumbing material may be useful to policymakers targeting schools for testing and remediation.

Introduction: The differentiation of Lewy body dementia from other common dementia types clinically is difficult, with a considerable number of cases only being found post-mortem. Consequently, there is a clear need for inexpensive and accurate diagnostic approaches for clinical use. Electroencephalography (EEG) is one potential candidate due to its relatively low cost and non-invasive nature. Previous studies examining the use of EEG as a dementia diagnostic have focussed on the eyes closed (EC) resting state; however, eyes open (EO) EEG may also be a useful adjunct to quantitative analysis due to clinical availability., Methods: We extracted spectral properties from EEG signals recorded under research study protocols (1024 Hz sampling rate, 10:5 EEG layout). The data stems from a total of 40 dementia patients with an average age of 74.42, 75.81 and 73.88 years for Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), respectively, and 15 healthy controls (HC) with an average age of 76.93 years. We utilised k-nearest neighbour, support vector machine and logistic regression machine learning to differentiate between groups utilising spectral data from the delta, theta, high theta, alpha and beta EEG bands., Results: We found that the combination of EC and EO resting state EEG data significantly increased inter-group classification accuracy compared to methods not using EO data. Secondly, we observed a distinct increase in the dominant frequency variance for HC between the EO and EC state, which was not observed within any dementia subgroup. For inter-group classification, we achieved a specificity of 0.87 and sensitivity of 0.92 for HC vs dementia classification and 0.75 specificity and 0.91 sensitivity for AD vs DLB classification, with a k-nearest neighbour machine learning model which outperformed other machine learning methods., Conclusions: The findings of our study indicate that the combination of both EC and EO quantitative EEG features improves overall classification accuracy when classifying dementia types in older age adults. In addition, we demonstrate that healthy controls display a definite change in dominant frequency variance between the EC and EO state. In future, a validation cohort should be utilised to further solidify these findings., (© 2022. The Author(s).)

Following the Flint Water Crisis, many states passed legislation requiring schools to measure and remediate lead in school drinking water. In this study, we present new evidence on the level and distribution of lead in school drinking water by examining the case of New York City, which tested water from every public school fixture in the 2016-17 school year, remediated fixtures that showed elevated levels of lead above 15 ppb, and retested a sample of fixtures in 2018-19. Prior to remediation, 8 % of fixtures showed elevated levels of lead; after remediation, 5 % of fixtures did. In both pre- and post-remediation periods, Black children attended schools with a higher proportion of elevated fixtures than White, Asian, and Hispanic children. We observe post-remediation lead exposure reductions that were largest for Black children, though racial disparities in exposure remained. Together, our results show that New York City's remediation efforts significantly reduced lead in its schools' drinking water in a short period of time, providing evidence of the promise of such efforts. However, the continued presence of lead in school drinking water and persistent racial disparities in exposure demonstrate the ongoing challenges to eradicating lead exposure in schools., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The 2015 American Thyroid Association endorsed less aggressive management for low-risk papillary thyroid cancer (LR-PTC). We aimed to identify factors influencing physicians' recommendations for LR-PTC., Methods: We surveyed members of three professional societies and assessed respondents' recommendations for managing LR-PTC using patient scenarios. Multivariable logistic regression models identified clinical and non-clinical factors associated with recommending total thyroidectomy (TT) and active surveillance (AS)., Results: The 345 respondents included 246 surgeons and 99 endocrinologists. Physicians' preference for their own management if diagnosed with LR-PTC had the strongest association with their recommendation for TT and AS (TT: OR 12.3; AS: OR 7.5, p < 0.001). Physician specialty and stated patient preference were also significantly associated with their recommendations for both management options. Respondents who received information about AS had increased odds of recommending AS., Conclusions: Physicians' recommendations for LR-PTC are strongly influenced by non-clinical factors, such as personal treatment preference and specialty., Competing Interests: Declaration of competing interest No competing financial interests exist., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The jail-based competency treatment (JBCT) model has become an established forensic practice across the country. From the perspective of implementation science and the three core elements of the Promoting Action on Research Implementation in Health Service (PARiHS) framework, the JBCT model is a remarkable example of how context (an unrelenting and overwhelmingly strong demand for forensic beds) has driven multiple state governments to facilitate implementation of a methodology in the absence of empirical evidence supporting its efficacy. This 7-year study of outcomes from four JBCT program sites provides this much-needed evidence by showing that JBCT restored 56% of 1553 male and 336 female patients over an average of 48.7 days. At the same time, the study highlights how variations in JBCT models, methods, and preadmission stabilization time present challenges to planned and effective implementation of evidence-based practice at the statewide system level. By identifying differential responsiveness to JBCT treatment by diagnosis and other factors, the study suggests preliminary implementation ideas for what types of patients are well served by the JBCT model as part of a continuum of restoration options that includes inpatient, outpatient and diversion. Significant findings showed that JBCT patients were restored at a higher rate and in a shorter time if they were female, < 20 years old (highest restoration rate; those < 60 years old also significantly better rates), free of co-occurring intellectual and cognitive deficits, and malingering. Of the major diagnoses, schizoaffective disorder required a significantly longer length of JBCT treatment for restoration, and lower restoration rates than schizophrenia and bipolar disorder, although this was moderated by a significant interaction with abuse of amphetamines., (© 2021 The Authors. Behavioral Sciences & The Law published by John Wiley & Sons Ltd.)

Objective: Active surveillance for low-risk papillary thyroid cancer (PTC) was endorsed by the American Thyroid Association guidelines in 2015. The attitudes and beliefs of physicians treating thyroid cancer regarding the active surveillance approach are not known., Methods: A national survey of endocrinologists and surgeons treating thyroid cancer was conducted from August to September 2017 via professional society emails. This mixed-methods analysis reported attitudes toward potential factors impacting decision-making regarding active surveillance, beliefs about barriers and facilitators of its use, and reasons why physicians would pick a given management strategy for themselves if they were diagnosed with a low-risk PTC. Survey items about attitudes and beliefs were derived from the Cabana model of barriers to guideline adherence and theoretical domains framework of behavior change., Results: Among 345 respondents, 324 (94%) agreed that active surveillance was appropriate for at least some patients, 81% agreed that active surveillance was at least somewhat underused, and 76% said that they would choose surgery for themselves if diagnosed with a PTC of ≤1 cm. Majority of the respondents believed that the guidelines supporting active surveillance were too vague and that the current supporting evidence was too weak. Malpractice and financial concerns were identified as additional barriers to offering active surveillance. The respondents endorsed improved information resources and evidence as possible facilitators to offering active surveillance., Conclusion: Although there is general support among physicians who treat low-risk PTC for the active surveillance approach, there is reluctance to offer it because of the lack of robust evidence, guidelines, and protocols., (Copyright © 2020 AACE. Published by Elsevier Inc. All rights reserved.)

Despite a continued evolution of the field of sexual abuser treatment toward a distinct professional discipline with clinicians using an increasing variety of treatment approaches, there is no consensus regarding the strength of our various clinical interventions as evidence-based practices (EBPs). This article provides a brief history of the development and goals of EBPs in medicine and mental health, and a review of the earnest efforts of researchers within the field to establish treatment approaches with sexual abusers as evidence-based. An appraisal of the current status of EBP's with sexual abusers is provided. Although there have been improvements in the methodological quality of treatment outcome research with sexual abusers, divergent opinions about treatment effectiveness remain, and the field has not yet agreed on a system or set of criteria for what constitutes "evidence." We contend that clinical practice has been influenced as much, or more, by new paradigms that are intuitively meaningful and perceived as needed than it has by what has been determined to be scientifically efficacious. This trend and other processes in our field that seem to be slowing the development of EBPs with sexual abusers are discussed. Recommendations for conducting evidence-based reviews and moving the field of sexual abuser treatment toward the use of a true EBP model are provided.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This review compiles 48 empirical studies and 55 clinical/practice articles specific to group therapy with sex offenders. Historically, group therapy has always been the predominant modality in sex offender-specific treatment. In the first decades of the field, treatment applied a psychoanalytic methodology that, although not empirically supported, fully appreciated the primary therapeutic importance of the group modality. Conversely, since the early 1980s, treatment has applied a cognitive behavioral method, but the field has largely neglected the therapeutic value of interpersonal group dynamics. The past decade has seen a growing re-appreciation of general therapeutic processes and more holistic approaches in sex offender treatment, and there is an emerging body of empirical research which, although often indirectly concerned with group, has yielded three definitive conclusions. First, the therapeutic qualities of the group therapist-specifically warmth, empathy, encouragement, and guidance-can strongly affect outcomes. Second, the quality of group cohesion can profoundly affect the effectiveness of treatment. Third, confrontational approaches in group therapy are ineffective, if not counter-therapeutic, and overwhelmingly rated as not helpful by sex offenders themselves. Additional conclusions are less strongly supported, but include compelling evidence that sex offenders generally prefer group therapy over individual therapy, that group therapy appears equally effective to individual therapy, and that mixing or separating groups by offense type is not important to therapeutic climate. Other group techniques and approaches specific to sexual abuse treatment are also summarized.

Objective: To describe the outcome of full-thickness skin grafts used to close skin defects involving the distal aspects of the limbs in cats and dogs and identify factors associated with outcome., Design: Retrospective case series., Animals: 20 cats and 32 dogs with a skin defect involving the distal aspect of a limb that received 58 full-thickness skin grafts between 2005 and 2012., Procedures: Data regarding patient signalment, location and cause of the skin defect, surgical and anesthetic duration, and postoperative bandaging protocol were obtained from the medical records. Graft outcome was assessed by interpreting descriptions in the records; skin viability over ≥ 75% of the graft area between 7 and 14 days after surgery was considered a successful outcome., Results: For 4 of the 58 grafts, graft outcome could not be determined from the medical record. For the remaining grafts, success rate was significantly higher for grafts placed in cats (17/22 [77%]) than in dogs (12/32 [38%]). The overall complication rate was 50%; complications included skin graft failure, donor site dehiscence, and bandage-induced sloughing of skin adjacent to the graft recipient site. In addition to species, anatomic location of the skin defect was identified as a prognostic indicator of graft outcome., Conclusions and Clinical Relevance: Full-thickness skin grafting had a higher success rate in cats than in dogs. Skin grafts applied to the antebrachium, compared with other locations on the distal aspects of the limbs, were associated with a poorer prognosis.

In 29 months of operation, the restoration of competency (ROC) program provided treatment services to 192 incompetent to stand trial patients in a jail setting. The ROC restored competency for 55% of the patients in an average of 57 days compared to the state hospital average of 180 days. The average cost of treatment/restoration per admission was $15,568 compared to the state hospital average of $81,000. The ROC model accelerates needed treatment for mentally ill defendants, cuts demand for costly state hospital forensic beds, and assists jails in better managing inmates with severe psychiatric disorders--yielding major cost savings and improved care. In addition to preventing readmissions and negative behavioral episodes, the ROC improved the broader forensic system by eliminating the state hospital waiting list, accelerating access to psychiatric services, promoting local access for lawyers and family, and gaining stakeholder satisfaction.

A national healthcare company applied the Malcolm Baldrige Criteria for Performance Excellence and its "Are We Making Progress?" survey as an annual organizational self-assessment to identify areas for improvement. For 6 years, Liberty Healthcare Corporation reviewed the survey results on an annual basis to analyze positive and negative trends, monitor company progress toward targeted goals and develop new initiatives to address emerging areas for improvement. As such, the survey provided a simple and inexpensive methodology to gain useful information from employees at all levels and from multiple service sites and business sectors. In particular, it provided a valuable framework for assessing and improving the employees' commitment to the company's mission and values, high standards and ethics, quality of work, and customer satisfaction. The methodology also helped the company to incorporate the philosophy and principles of continuous quality improvement in a unified fashion. Corporate and local leadership used the same measure to evaluate the performance of individual programs relative to each other, to the company as a whole, and to the "best practices" standard of highly successful companies that received the Malcolm Baldrige National Quality Award., (© 2012 National Association for Healthcare Quality.)

Objectives: Research indicates an association between stimulating mental activities and better memory performance as people age, but studies on computerized mental stimulation programs are limited. We explored whether computerized brain training exercises improved cognitive performance in older adults., Methods: In local retirement communities, a convenience sample was randomized into an intervention group (N = 36), who used a computer program 5 days a week for 20-25 minutes each day, or a wait-list control group (N = 33). All were older adults without dementia (mean age: 81.8 years; SD: 6.1; 67% female). Neuropsychological testing was completed at baseline (Time 1), 2 months (Time 2), and 6 months (Time 3). Three cognitive domains (Immediate Memory, Delayed Memory, Language) were compared in the two groups as a function of time using mixed models., Results: The intervention group used the computerized program (Brain Fitness, Dakim Inc., Santa Monica, CA) for an average of 43 (SD: 4.4) sessions by Time 2 and 81 (SD: 37.5) sessions by Time 3. Mixed models examining cognitive domains as function of time revealed significant group differences in Delayed Memory (F(2,72) = 4.7, p = 0.01) but not Immediate Memory and Language; no significant improvements were noted for the control group. Among all participants, anyone playing at least 40 sessions over the 6 months improved in all three domains (Immediate Memory, Delayed Memory, and Language)., Conclusion: Participating in a computerized brain exercise program over 6 months improves cognitive abilities in older adults. These results extend literature indicating the benefit of training exercises, whether in a classroom format or via a computerized self-paced program., (Copyright © 2013 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.)

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

Though many studies have suggested that social and behavioral skills play a central role in gender stratification processes, we know little about the extent to which these skills affect gender gaps in academic achievement. Analyzing data from the Early Child Longitudinal Study-Kindergarten Cohort, we demonstrate that social and behavioral skills have substantively important effects on academic outcomes from kindergarten through fifth grade. Gender differences in the acquisition of these skills, moreover, explain a considerable fraction of the gender gap in academic outcomes during early elementary school. Boys get roughly the same academic return to social and behavioral skills as their female peers, but girls begin school with more advanced social and behavioral skills and their skill advantage grows over time. While part of the effect may reflect an evaluation process that rewards students who better conform to school norms, our results imply that the acquisition of social and behavioral skills enhances learning as well. Our results call for a reconsideration of the family and school-level processes that produce gender gaps in social and behavioral skills and the advantages they confer for academic and later success., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Vibrionic hepatitis is a disease of poultry which is characterised by the presence of focal lesions in the liver, usually 1-2mm in size and greyish-white in colour. The cause of the disease remains unclear, as do the reasons for its recent re-emergence. We examined the livers of commercial broiler chickens taken during processing and found Campylobacter spp. in both normal livers and those displaying signs indicative of focal hepatitis. Livers with signs of hepatitis had significantly more Campylobacter spp. present than those without and other bacterial genera were infrequently present. We were unable to replicate the disease in a healthy host following experimental infection with a Campylobacter jejuni strain isolated from a liver showing signs of focal hepatitis. However, a significant T cell response to C. jejuni was seen in the liver of Campylobacter infected birds. We conclude that the presence of Campylobacter spp. in the liver alone is not sufficient to cause vibrionic hepatitis, but that a predisposing factor, possibly within the host is required. We also provide evidence that chickens mount an adaptive T cell response to systemic C. jejuni., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC-MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. In Vivo metabolic labeling of proteins with (32)P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.

This study presents the long-term outcomes of a continuum of care program for co-occurring psychiatric disabilities and chemical dependency that has been recognized as a best practice model by the American Psychological Association's Committee for the Advancement of Professional Practice's Task Force on Serious Mental Illness and Severe Emotional Disturbance (APA/CAPP, 2007). Since publication of the initial positive outcomes for 18 men in 2002, this innovative recovery program continued to successfully reintegrate a total of 91 men and women with severe co-occurring disabilities who had been acquitted of violent crimes by reason of insanity (NGRI). This follow-up study showed continued positive outcomes for an additional 73 program graduates in terms of non-reoffending, psychiatric stability, substance abuse abstinence, stable housing and meaningful activity. In contrast to other studies that have applied Assertive Community Treatment and Intensive Case Management to populations with forensic issues and failed to reduce criminal recidivism, this continuum of care recovery model had strong results in preventing criminal recidivism in addition to achieving improved mental health, abstinence and quality of life.

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

Yeast Mot1p, a member of the Snf2 ATPase family of proteins, is a transcriptional regulator that has the unusual ability to both repress and activate mRNA gene transcription. To identify interactions with other proteins that may assist Mot1p in its regulatory processes, Mot1p was purified from replicate yeast cell extracts, and Mot1p-associated proteins were identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. Using this approach we generated a catalog of Mot1p-interacting proteins. Mot1p interacts with a range of transcriptional co-regulators as well as proteins involved in chromatin remodeling. We propose that interaction with such a wide range of proteins may be one mechanism through which Mot1p subserves its roles as a transcriptional activator and repressor.

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men.

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. The APC/C becomes active at the metaphase/anaphase transition and remains active during G(1) phase. One mechanism linked to activation of the APC/C is phosphorylation. Although many sites of mitotic phosphorylation have been identified in core components of the APC/C, the consequence of any individual phosphorylation event has not been elucidated in vivo. In this study, we show that Hcn1 is an essential core component of the fission yeast APC/C and is critical for maintaining complex integrity. Moreover, Hcn1 is a phosphoprotein in vivo. Phosphorylation of Hcn1 occurs at a single Cdk1 site in vitro and in vivo. Mutation of this site to alanine, but not aspartic acid, compromises APC/C function and leads to a specific defect in the completion of cell division.

Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.

Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

Septins are GTP binding proteins important for cytokinesis in many eukaryotes. The Schizosaccaromyces pombe genome sequence predicts orthologues of four of five Saccharomyces cerevisiae septins involved in cytokinesis and these are named Spns1-4p. That spns1-4 are not essential genes permitted the application of a combined genetic and proteomics approach to determine their functional relationships. Our findings indicate that Spns1-4p are present throughout interphase as a diffusely localized approximately 8.5S complex containing two copies of each septin linked together as a chain in the order Spn3p-Spn4p-Spn1p-Spn2p. Septin recruitment to the medial region of the cell is genetically separable from ring formation, and whereas it is normally restricted to mitosis, it can be promoted without activation of the mitotic cell cycle machinery. Coalescence into ring structures requires Spn1p and Spn4p associate with at least one other septin subunit and the expression of Mid2p that is normally restricted to mitosis. This study establishes the functional requirements for septin complex organization in vivo.

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions., Competing Interests: The authors have declared that no conflicts of interest exist.

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.

Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution.

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

Group-based interventions are widely used as the preferred method for the treatment of sexual offenders. However, little attention has been given to the vital interpersonal processes within groups, which contribute to their therapeutic effect. This paper describes specific principles and techniques of group therapy that will help sex offender treatment practitioners take full advantage of this unique modality and thereby maximize the positive impact of treatment. In contrast to an individual-focused style that characterizes many sex offender treatment groups, group-focused interventions involve all group members and use the social/relational energy that is essential to the group modality.

The positive transcriptional elongation factor b (P-TEFb), consisting of CDK9 and cyclin T, stimulates transcription by phosphorylating RNA polymerase II. It becomes inactivated when associated with the abundant 7SK snRNA. Here, we show that the 7SK binding alone was not sufficient to inhibit P-TEFb. P-TEFb was inhibited by the HEXIM1 protein in a process that specifically required 7SK for mediating the HEXIM1:P-TEFb interaction. This allowed HEXIM1 to inhibit transcription both in vivo and in vitro. P-TEFb dissociated from HEXIM1 and 7SK in cells undergoing stress response, increasing the level of active P-TEFb for stress-induced transcription. P-TEFb was the predominant HEXIM1-associated protein factor, and thus likely to be the principal target of inhibition coordinated by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells treated with hexamethylene bisacetamide, a potent inducer of cell differentiation, targeting the general transcription factor P-TEFb by HEXIM1/7SK may contribute to the global control of cell growth and differentiation.

The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. Components of the APC have been identified through genetic screens in both Schizosaccharomyces pombe and Saccharomyces cerevisiae as well as through biochemical purification coupled with mass spectrometric protein identification. With these approaches, 11 subunits of the core S. cerevisiae APC have been identified. Here, we have applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S. pombe and S. cerevisiae APCs. Our data increase the total number of identified APC subunits to 13 in both yeasts and indicate that previous approaches were biased against the identification of small subunits. These results underscore the power of direct analysis of protein complexes by mass spectrometry and set the foundation for further functional and structural studies of the APC.

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.

Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.