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Background: To assess outcomes of health services, providers need brief, responsive, reliable, and valid measures that can be implemented in clinical settings with minimal cost and burden. The Behavior and Symptom Identification Scale (BASIS-32) is a self-report measure developed in 1984 to assess mental health treatment outcomes. During the past 3 years, multiple methods were used to revise the instrument to improve reliability, validity, and applicability to diverse groups of mental health service recipients.Objective: The objective of this study was to field test the revised instrument, make further changes based on analysis of the field test data, and assess reliability and validity of the final version (BASIS-24).Methods: A field test was implemented at 27 treatment sites across the United States. A total of 2656 inpatients and 3222 outpatients participated. Factor analytic methods, classic test theory, and item response theory modeling were used to select the most discriminating, nonredundant items for inclusion in the final version of the instrument and to assess its reliability and validity. Item response theory modeling was used to score the instrument.Results: The final instrument includes 24 items assessing 6 domains: depression/ functioning, interpersonal relationships, self-harm, emotional lability, psychosis, and substance abuse. Test-retest and internal consistency reliability were acceptable. Tests of construct and discriminant validity supported the instrument's ability to discriminate groups expected to differ in mental health status, and its correlation with other measures of mental health.Conclusions: Analyses of the BASIS-24 supported its reliability and validity for assessing mental health status from the patient's perspective. [ABSTRACT FROM AUTHOR]

Objective: In 2006, the field of stem cell research was captivated by a possibility of epigenetic reprogramming of terminally differentiated cells to a pluripotent state by four factors - Oct4, Sox2, Klf4 and c-Myc. Hovewer, six years after this breakthrough, we still lack proper insight into this fascinating process on a molecular level. Among all four factors in the original cocktail, Oct4 evinces a special feature as it is impossible to replace this transcription factor by any other member of its protein family. But what makes the protein so unique? Materials and Methods: Purification and assembly of the Oct4POU:PORE complex; Crystallization and structure determination; Electrophoretic mobility shift assay; Reprogramming and rescue assay; Strep-tagged purification of proteins; Mass spectrometry; Yeast two hybrid screen; Genetic code expansion technology. Results: We solved the crystal structure of Oct4. This revealed a unique structured linker sequence of Oct4, which is not found in other POU class members. Moreover, this alpha-helical stretch is exposed to surface, creating an attractive interface regarding direct interactions with other components of the reprogramming machinery. A mutation screen along the linker proved several amino acids to play a crucial role in reprogramming. Based on current interactome studies, we conclude that the region is involved in recruiting key epigenetic players. Two different approaches have been chosen to determine the direct interaction partners of aforesaid Oct4 alpha helix. The results of a yeast two hybrid screen and in vivo crosslinking via the genetic code expansion technology will be presented. Conclusion: The linker region of Oct4 is involved in recruiting key epigenetic players during the reprogramming process. [ABSTRACT FROM AUTHOR]

The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.

BAF chromatin remodeling complexes containing the BRG1 protein have been shown to be not only essential for early embryonic development, but also paramount in enhancing the efficiency of reprogramming somatic cells to pluripotency mediated by four transcription factors. To investigate the role of BRG1 in regulating pluripotency, we found that Oct4 and Nanog levels were increased immediately after BRG1 knockdown. While Nanog levels remained elevated over the investigated time period, Oct4 levels decreased at later time points. Additionally, OCT4 target genes were also found to be upregulated upon Brg1 knockdown. SiRNA-mediated BRG1 knockdown in embryonic stem (ES) cells led to Oct4 and Nanog upregulation, whereas F9 cells showed primarily Oct4 upregulation. BRG1 knockdown upregulated the expression of differentiation markers in mouse ES cells as well as differentiated morphology under reduced leukemia inhibitory factor conditions. Our results show that BRG1 plays an important role in maintaining pluripotency by fine-tuning the expression level of Oct4 and other pluripotency-associated genes.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein's surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein-protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process.

The recent association of certain influenza A virus subtypes with clinically relevant phenotypes has led to the increasing importance of subtyping by clinical virology laboratories. To provide clinical laboratories with a definitive immunofluorescence assay for the subtyping of influenza A virus isolates, we generated a panel of monoclonal antibodies (MAbs) against the major circulating influenza A virus subtypes using multiple inactivated H1N1, H3N2, and 2009 H1N1 strains individually as immunogens. Eleven MAbs that target hemagglutinin (HA) of H1N1 and H3N2 subtypes were selected. These MAbs were combined into three subtype-specific reagents, one each for pan-H1 (seasonal and 2009 strains), H3, and 2009 H1, for the subtyping of influenza A virus-positive specimens by indirect immunofluorescence assay (IFA). Each subtype-specific reagent was tested on 21 prototype influenza A virus strains and confirmed to be specific for its intended subtype. In addition, the subtyping reagents did not cross-react with any of 40 other viruses. The clinical performance of the subtyping reagents was evaluated with 75 archived clinical samples collected between 2006 and 2009 using the D(3) Ultra DFA influenza A virus identification reagent (Diagnostic Hybrids, Inc., Athens, OH) and the influenza A virus subtyping reagents by IFA simultaneously. Sixty-four samples grew virus and were subtyped as follows: 30 as H3N2, 9 as seasonal H1N1, and 25 as 2009 H1N1. RT-PCR was used to confirm the influenza A virus subtyping of these samples, and there was 100% agreement with IFA. This subtyping IFA provides clinical laboratories with a cost-effective diagnostic tool for better management of influenza virus infection and surveillance of influenza virus activity.

Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs). However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs), indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296) led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4), and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells.

The evolutionary origins of the gene network underlying cellular pluripotency, a central theme in developmental biology, have yet to be elucidated. In mammals, Oct4 is a factor crucial in the reprogramming of differentiated cells into induced pluripotent stem cells. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Here we report that different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts and that the inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent cells. Therefore, our results indicate that induction of pluripotency is not a feature specific to mammals, but existed in the Oct4/Pou2 common ancestral vertebrate.

Background: The prediction of transcription factor binding sites is difficult for many reasons. Thus, filtering methods are needed to enrich for biologically relevant (true positive) matches in the large amount of computational predictions that are frequently generated from promoter sequences., Results: ReXSpecies 2 filters predictions of transcription factor binding sites and generates a set of figures displaying them in evolutionary context. More specifically, it uses position specific scoring matrices to search for motifs that specify transcription factor binding sites. It removes redundant matches and filters the remaining matches by the phylogenetic group that the matrices belong to. It then identifies potential transcriptional modules, and generates figures that highlight such modules, taking evolution into consideration. Module formation, scoring by evolutionary criteria and visual clues reduce the amount of predictions to a manageable scale. Identification of transcription factor binding sites of particular functional importance is left to expert filtering. ReXSpecies 2 interacts with genome browsers to enable scientists to filter predictions together with other sequence-related data., Conclusions: Based on ReXSpecies 2, we derive plausible hypotheses about the regulation of pluripotency. Our tool is designed to analyze transcription factor binding site predictions considering their common pattern of occurrence, highlighting their evolutionary history.

Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

Increasing racial and ethnic diversity calls for mental health assessment instruments that are appropriate, reliable, and valid for the wide range of cultures that comprise the current US population. However, most assessment instruments have not been tested on diverse samples. This study assessed psychometric properties and sensitivity to change of the revised Behavior and Symptom Identification Scale (BASIS-24) among the three largest race/ethnicity groups in the USA: Whites, African-Americans, and Latinos. BASIS-24 assessments were obtained for 2436 inpatients and 2975 outpatients treated at one of 27 mental health and/or substance abuse programs. Confirmatory factor analysis and several psychometric tests supported the factor structure, reliability, concurrent validity, and sensitivity of the instrument within each race/ethnicity group, although discriminant validity may be weaker for African-Americans and Latinos than for Whites. Further research is needed to test and validate assessment instruments with other race/ethnicity groups.

Objectives: To examine patterns of computer and Internet use among persons with spinal cord injuries (SCI) and to assess the relationship between Internet use and health-related quality of life (HRQOL)., Design: Cross-sectional survey design., Setting: National Model Spinal Cord Injury Systems., Participants: People with SCI enrolled in a national database., Interventions: Not applicable., Main Outcome Measures: Patterns of Internet use and relationship to HRQOL indicators: self-perceived health status, health status compared with 1 year ago, severity of depression, social integration score, occupation score, contacts with friends, business contacts, and satisfaction with life., Results: Most subjects owned computers, had Internet access, and used the Internet regularly-primarily for email, disability and health information, and shopping. Bivariate analysis revealed significant differences in Internet access based on sociodemographics, particularly among subjects with less education and among African Americans and Hispanics. In initial univariate analysis, most HRQOL indicators were significantly better for Internet users; once sociodemographic factors were included, 4 indicators remained significant., Conclusions: Complex factors contribute to Internet access among people with SCI, with more barriers among specific subgroups. A significant HRQOL benefit from Internet use is suggested. Targeted interventions and studies of usage patterns are recommended.