Cross-linking of DNA through HMGA1 suggests a DNA scaffold.

Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.

A bisexually reproducing all-triploid vertebrate.

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community. The reproduction of naturally occurring triploids has been reported to occur only through parthenogenesis, gynogenesis or hybridogenesis. The bisexual reproduction of pure triploids has been considered to be impossible because of the problem of equally distributing three chromosome sets in meiosis. Here we report geographically isolated populations of green toads (Bufo viridis complex) that are all-triploid and reproduce bisexually.

Health care access and treatment for children with co-morbid autism and psychiatric conditions.

PURPOSE: To characterize the rate of comorbid psychiatric conditions (CPC) among children with autism spectrum disorders (ASD), to examine their treatment utilization, and to investigate treatment delay or non-delivery. METHODS: Lifetime ASD and CPC in children, aged 2-17, were investigated using data from the 2007-2008 National Survey of Children's Health (NSCH). The NSCH surveyed parents and guardians regarding the health and well being, including treatment, of their child(ren) under age 18 (n = 91,642). Children with health conditions were defined by parent report that a doctor or other health professional had ever said their child had that condition. Factors related to overall health, treatment utilization, and barriers to access variables were investigated among this group. RESULTS: Children with ASD/CPC had poorer overall health outcomes than children with ASD alone. They more often were dissatisfied with their between-provider communication and less often had insurance cover needed services. Nonetheless, they did tend to use care coordination and mental health services to a greater degree. Families were more likely to report the delay or non-receipt of needed services when they perceived a lack of communication and partnership with providers, when they lacked insurance coverage, and when they felt that health care costs were unreasonable. CONCLUSIONS: The presence of a CPC seems to shape the treatment utilization and health outcomes of children with ASD. Because of this, health professionals working with children with autism should give special attention to treatment of those with comorbid diagnoses.

Advances in Supporting Parents in Interventions for Autism Spectrum Disorder.

The rising prevalence of autism spectrum disorder (ASD) calls for clear referral and treatment guidelines for children with ASD and their caregivers. Caregiver involvement in intervention is a standard practice of care and research suggests that teaching intervention strategies to caregivers can improve child outcomes and increase caregiver efficacy. Caregiver-mediated interventions that are naturalistic, developmental, and behavioral are effective in improving social and communication skills for children with ASD. Caregiver training models that use behavioral strategies are effective in reducing challenging behaviors. Finally, reducing caregiver barriers to treatment implementation, including stress and strain, are becoming critical components for improving the well-being and care of children with ASD and their families.

Binding and interplay of HMG proteins on chromatin: lessons from live cell imaging.

Members of the superfamily of high mobility group (HMG) proteins are considered as architectural elements of chromatin. It is now clear that they belong to a network of dynamic chromatin proteins that constantly move around the chromatin fiber thereby dynamically modulating DNA-dependent processes. In this review we discuss how HMGs fused to fluorescent proteins and live cell imaging advanced our understanding in HMG dynamics and function. By presenting the regulation of the dynamic properties of each HMG family in comparison to one another we wish to highlight common themes among the three families, as well as stimulate new ideas from one HMG family in relation to the others and more generally in the dynamic world of chromatin.

Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins.

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G(1) and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation.

BACKGROUND: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. RESULTS: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. CONCLUSIONS: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells.

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

Comparing Neighborhoods of Children With Autism Spectrum Disorder in a Medicaid Waiver Program and a State Population, 2007-2015.

OBJECTIVE: This study investigated equity in enrollment in a Medicaid waiver program for early intensive behavioral intervention for children with autism spectrum disorder (ASD). METHODS: State administrative, Medicaid, and U.S. Census data for children enrolled in the waiver program between 2007 and 2015 (N=2,111) were integrated. Multivariate and bivariate analyses were used to compare enrollees' neighborhood demographic characteristics with those of the state's general population, with controls for enrollees' age, sex, and race-ethnicity. RESULTS: Findings indicate that in general, enrollment was equitable. During the years in which there were inequities, children who lived in neighborhoods of privilege were favored. These neighborhoods had higher median incomes, lower poverty levels, and fewer female-headed households and were located in urban areas. CONCLUSIONS: As states work to provide equitable treatment to children with ASD and their families, it is important to track potential inequities between children who do and do not enroll in services and to use this information to inform outreach efforts. States may turn to South Carolina for insight on how to ensure equity.

Treatment Utilization Trajectories among Children with Autism Spectrum Disorder: Differences by Race-Ethnicity and Neighborhood.

Health coverage of early intensive behavioral intervention (EIBI) for children with autism spectrum disorder is expanding. Yet there is no longitudinal research on patterns of or inequities in utilization of EIBI. We integrated state administrative records with Medicaid and Census data for children enrolled in an EIBI Medicaid waiver (N = 730) to identify and describe the type and prevalence of treatment utilization trajectories, and to examine the association between trajectory types and (a) child race-ethnicity and (b) neighborhood racial composition, poverty, affluence, and urbanicity. We identified four utilization trajectories (Low, Low-Moderate, Moderate, and High users). Race-ethnicity and neighborhood affluence were associated with trajectory membership. As coverage expands, policy makers should consider strategies to improve overall treatment utilization and enhance equity.

S-adenosyl-L-methionine counteracts mitotic disturbances and cytostatic effects induced by sodium arsenite in HeLa cells.

Aneuploidy represents a serious problem for human health. Toxicological data have shown that aneuploidy can be caused by exposure to chemical agents known as mitotic spindle poisons, since they arrest cell cycle in mitosis through their interaction with tubulin. Among these agents is arsenic. In previous reports, we demonstrated that the aneugenic events induced by sodium arsenite can be abolished by the exogenous addition of S-adenosyl-l-methionine (SAM). Nevertheless, the mechanisms involved are still unknown. The aim of the present work was to study the influence of SAM on the mitotic disturbances caused by sodium arsenite. To achieve this goal, we analyzed microtubule (MT) polymerization by immunolocalization and live cell microscopy of mitotic cells. Our findings indicate that sodium arsenite alters the dynamics of MT polymerization, induces centrosome amplification and delays mitosis. Furthermore, SAM reduces the alterations on MT dynamics, as well as centrosome amplification, and therefore diminishes the formation of multipolar spindles in treated HeLa cells. In addition, SAM decreases the progression time through mitosis. Taking these data together, we consider that the mechanism by which SAM reduces the frequency of aneuploid cells must be related to the modulation of the dynamics and organization of MT, suggesting a role of SAM on chromosome segregation, which should be further investigated in primary cells.

Time-Lag Between Diagnosis of Autism Spectrum Disorder and Onset of Publicly-Funded Early Intensive Behavioral Intervention: Do Race-Ethnicity and Neighborhood Matter?

Health coverage of early intensive behavioral intervention (EIBI) for children with autism spectrum disorder (ASD) is rapidly expanding across the United States. Yet we know little about the time-lag between diagnosis and treatment onset. We integrated administrative, Medicaid claims, and Census data for children in an EIBI Medicaid waiver (n = 473) to examine the relationship between time-lag and (a) child race-ethnicity and (b) neighborhood racial composition, poverty, affluence, and urbanicity. We explored whether the relationship between child race-ethnicity and time-lag varies by neighborhood characteristics. Average time-lag between diagnosis and treatment onset was nearly 3 years. Child race-ethnicity and neighborhood characteristics did not predict time-lag. Reducing time-lag is critical to ensuring that children with ASD receive treatment as early as possible.

Mitotic phosphorylation of chromosomal protein HMGN1 inhibits nuclear import and promotes interaction with 14.3.3 proteins.

Progression through mitosis is associated with reversible phosphorylation of many nuclear proteins including that of the high-mobility group N (HMGN) nucleosomal binding protein family. Here we use immunofluorescence and in vitro nuclear import studies to demonstrate that mitotic phosphorylation of the nucleosomal binding domain (NBD) of the HMGN1 protein prevents its reentry into the newly formed nucleus in late telophase. By microinjecting wild-type and mutant proteins into the cytoplasm of HeLa cells and expressing these proteins in HmgN1(-/-) cells, we demonstrate that the inability to enter the nucleus is a consequence of phosphorylation and is not due to the presence of negative charges. Using affinity chromatography with recombinant proteins and nuclear extracts prepared from logarithmically growing or mitotically arrested cells, we demonstrate that phosphorylation of the NBD of HMGN1 promotes interaction with specific 14.3.3 isotypes. We conclude that mitotic phosphorylation of HMGN1 protein promotes interaction with 14.3.3 proteins and suggest that this interaction impedes the reentry of the proteins into the nucleus during telophase. Taken together with the results of previous studies, our results suggest a dual role for mitotic phosphorylation of HMGN1: abolishment of chromatin binding and inhibition of nuclear import.

Prevention of aneuploidy by S-adenosyl-methionine in human cells treated with sodium arsenite.

Alterations in methyl group's metabolism affect availability of S-adenosyl-L-methionine (SAM); these modifications can be originated by enzyme polymorphisms, nutritional deficiencies, and exposure to chemical agents. There are several types of chemicals that interfere with methyl groups, among them is arsenic. It deserves special attention because it modifies a number of cell functions that influence the development of diseases such as cancer. Since part of arsenic's toxicity is influenced by changes on SAM availability, in a previous study we investigated whether exogenous addition of SAM to cells treated with sodium arsenite (NaAsO(2)) has an effect on its genotoxicity. Results demonstrated that SAM reduces the frequency of cells presenting micronuclei (MN) and tubulin-cytoskeleton defects after treatment with NaAsO(2). MN are fragments of the cell nucleus that may contain whole chromosomes or chromosome fragments depending on whether they derive either from the aneugenic or from the clastogenic action of chemicals. Therefore one question generated by these results was whether SAM reduced only the frequency MN resulting from aneugenic damage. To answer this question, in the present work we used an all-centromere DNA probe to distinguish the type of MN reduced by SAM after treatment with NaAsO(2) and vinblastine. In addition, the capacity of SAM to reduce clastogenicity was also evaluated. Results show that SAM decreases the frequency of cells with MN containing whole chromosomes in cultures treated either with NaAsO(2) or with vinblastine; however, induction of double-strand breaks by NaAsO(2) was not prevented by SAM.

Parent perceived challenges to treatment utilization in a publicly funded early intensive behavioral intervention program for children with autism spectrum disorder.

Background: In recent years, the delivery of early intensive behavioral intervention (EIBI) for children with autism spectrum disorder (ASD) in the United States has significantly changed. More children with ASD than ever before are eligible to use publicly funded EIBI. Yet, the challenges to large-scale implementation of EIBI remain unclear. Specific Aims: We examined parent perceived challenges to treatment utilization, predictors of increased challenges to treatment utilization, and parent recommendations for increasing utilization in a statewide EIBI program. Method: Using a cross-sectional design, we surveyed parents of children with ASD receiving EIBI through South Carolina's Pervasive Developmental Disorder Program (N = 145). To examine the contributions of parent demographic characteristics, parent social support, and child challenging behaviors to perceived challenges to utilization, we used multiple linear regression. Parent recommendations were collected using a single open-ended question. Findings: The most frequently endorsed challenges included the child's school schedule (62.1%) and the child being overburdened with other treatment demands (65.2%). Greater child challenging behaviors were associated with a greater degree of perceived challenges, and social support was associated with a lesser degree of perceived challenges. Discussion: Parents perceived various challenges to utilization, and child and family characteristics may increase the risk for experiencing challenges to utilization. As the delivery of EIBI continues to evolve in the United States and elsewhere, these findings have implications for policy, programming, and future research.

Expression of HMGA2 variants during oogenesis and early embryogenesis of Xenopus laevis.

The high mobility group proteins A2 (HMGA2) have been implicated in the control of cell proliferation and differentiation, in particular during embryogenesis. Here, we used Xenopus laevis to analyze HMGA2 gene expression patterns during oogenesis and early embryogenesis. We found two functional XlHMGA2 isoforms, which we named XlHMGA2alpha and XlHMGA2beta. As revealed by RT-PCR, real-time PCR and whole-mount in situ hybridization both mRNAs are maternally produced and stored in eggs. Whole-mount in situ hybridizations revealed a conspicuous redistribution of the XlHMGA2 transcripts during early embryogenesis. Initially, during oogenesis and in eggs, the transcripts are uniformly distributed in the cytoplasm. With activation of the eggs the transcripts accumulate near the animal pole and remain in the juxtanuclear regions of animal pole blastomeres until midblastula transition. According to real-time PCR data, XlHMGA2alpha appears to be preferentially expressed during oogenesis and after midblastula transition, whereas XlHMGA2beta expression predominates after neurulation, suggesting an individual transcriptional regulation.

Barriers and Facilitators to Substance Use Treatment in the Rural South: A Qualitative Study.

PURPOSE: Little qualitative research has examined factors associated with care in substance abuse treatment agencies in Southeastern rural communities. This study explored client- and agency stakeholder-perceived barriers and facilitators to substance use treatment delivery in southeastern rural communities. METHODS: Group and individual interviews were conducted with 40 key stakeholders and 40 clients at 9 substance abuse agencies serving rural communities in a southeastern state. Qualitative thematic analysis was used to identify perceived barriers and facilitators to substance abuse services in rural communities. FINDINGS: Four primary themes emerged from the client and stakeholder interviews as both barriers and facilitators: availability of services for individuals with substance use disorders; access to the current technology for client services and agency functioning; cost of services; and stigma. CONCLUSIONS: This study identifies novel barriers and facilitators to substance use care in the rural South and highlights essential areas for consideration when developing and implementing substance use care in this geographic region. These findings can be used as guidelines to provide better care to individuals with substance use disorders living in rural communities.

Developmental role of HMGN proteins in Xenopus laevis.

HMGN proteins are architectural chromatin proteins that reduce the compaction of the chromatin fiber, facilitate access to nucleosomes and modulate replication and transcription processes. Here we demonstrate that in Xenopus laevis, the expression and cellular location of the HMGN proteins are developmentally regulated and that their misexpression leads to gross developmental defects in post-blastula embryos. HMGN transcripts and proteins are present throughout oogenesis; however, the proteins stored in the cytoplasm are not associated with lampbrush chromosomes, and are rapidly degraded when oocytes mature into eggs. During embryogenesis, HMGN expression is first detected in blastula stages and progresses to a tissue-specific expression reaching relative high levels in the mesodermal and neuroectodermal regions of tadpoles. Only after midblastula transition (MBT), alterations in the HMGN levels by either microinjection of recombinant proteins or by morpholino-antisense oligo treatments produced embryos with imperfectly closed blastopore, distorted body axis and showed abnormal head structures. Analyses of animal cap explants indicated that HMGN proteins are involved in the regulation of mesoderm specific genes. In addition, HMGN misexpression caused altered expression of specific genes at MBT rather than global changes of transcription rates. Our results demonstrate that proper embryonic development of Xenopus laevis requires precisely regulated levels of HMGN proteins and suggest that these nucleosomal binding proteins modulate the expression of specific genes.

Examining treatment adherence among parents of children with autism spectrum disorder.

BACKGROUND: Children with Autism Spectrum Disorder (ASD) participate in a variety of treatments, including medication, behavioral, alternative and developmental treatments. Parent adherence to these treatments is crucial for positive child outcomes. OBJECTIVE: The current study: 1) Explored patterns of parent adherence across the full range of treatments that are prescribed to children with ASD and, 2) Examined whether parent demographics, parent treatment attitudes, and child ASD severity contribute to parents' adherence across ASD treatments. METHOD: Questionnaires were distributed to parents of children with ASD in a southeastern state. Parents (N = 274) were included if they were parenting a child with ASD who was receiving treatment for ASD symptoms. Paired t-tests and multiple linear regression were used to assess the study aims. RESULTS: Adherence to medication treatment was significantly greater than adherence to behavioral, developmental, or alternative treatments (adjusted p-values 0.0006, 0.0030, 0.0006 respectively). Perceived family burden of a treatment was associated with lower adherence to medication, developmental, and alternative treatments. Finally, greater ASD severity was associated with lower adherence to alternative treatments. CONCLUSION: Overall, the independent variables accounted for more variance in adherence to medication and alternative treatments than in behavioral and developmental treatments. Parents' adherence to ASD treatment differs significantly by treatment type and is influenced by parental perceptions of the burden of treatment on the family. These findings highlight the importance of understanding and addressing the impact of ASD treatment regimens on family life.