The Role of Dot1l in Prenatal and Postnatal Murine Chondrocytes and Trabecular Bone.

Osteoarthritis and osteoporosis are widely prevalent and have far-reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase DOT1L, plays an important role in the cartilage and bone biology. In this study, we evaluated the role of Dot1l in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte-specific constitutive and inducible conditional Dot1l KO mouse lines using Col2a1-Cre and Acan-CreER systems. Prenatal deletion of Dot1l in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of Col10a1 expression. Postnatal deletion of Dot1l in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, Dot1l is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.

Glial cell responses in a murine multifactorial perinatal brain injury model.

The impact of traumatic brain injury during the perinatal period, which coincides with glial cell (astrocyte and oligodendrocyte) maturation was assessed to determine whether a second insult, e.g., increased inflammation due to remote bacterial exposure, exacerbates the initial injury's effects, possibly eliciting longer-term brain damage. Thus, a murine multifactorial injury model incorporating both mechanisms consisting of perinatal penetrating traumatic brain injury, with or without intraperitoneal injection of lipopolysaccharide (LPS), an analog of remote pathogen exposure has been developed. Four days after injury, gene expression changes for different cell markers were assessed using mRNA in situ hybridization (ISH) and qPCR. Astrocytic marker mRNA levels increased in the stab-alone and stab-plus-LPS treated animals indicating reactive gliosis. Activated microglial/macrophage marker levels, increased in the ipsilateral sides of stab and stab-plus LPS animals by P10, but the differences resolved by P15. Ectopic expression of glial precursor and neural stem cell markers within the cortical injury site was observed by ISH, suggesting that existing precursors and neural stem cells migrate into the injured areas to replace the cells lost in the injury process. Furthermore, single exposure to LPS concomitant with acute stab injury affected the oligodendrocyte population in both the injured and contralateral uninjured side, indicating that after compromise of the blood-brain barrier integrity, oligodendrocytes become even more susceptible to inflammatory injury. This multifactorial approach should lead to a better understanding of the pathogenic sequelae observed as a consequence of perinatal brain insult/injury, caused by combinations of trauma, intrauterine infection, hypoxia and/or ischemia in humans.

Aggrecan is required for growth plate cytoarchitecture and differentiation.

The proteoglycan aggrecan is a prominent component of the extracellular matrix in growth plate cartilage. A naturally occurring, recessive, perinatally lethal mutation in the aggrecan core protein gene, cmd(bc) (Acan(cmd-Bc)), that deletes the entire protein-coding sequence provided a model in which to characterize the phenotypic and morphologic effects of aggrecan deletion on skeletal development. We also generated a novel transgenic mouse, Tg(COL2A1-ACAN), that has the chick ACAN coding sequence driven by the mouse COL2A1 promoter to enable the production of cmd(bc)/cmd(bc); Tg(COL2A1-ACAN) rescue embryos. These were used to assess the impact of aggrecan on growth plate organization, chondrocyte survival and proliferation, and the expression of mRNAs encoding chondrocyte differentiation markers and growth factors. Homozygous mutant (cmd(bc)/cmd(bc)) embryos exhibited severe defects in all skeletal elements with deformed and shortened (50%) limb elements. Expression of aggrecan in rescue embryos reversed the skeletal defects to varying degrees with a 20% increase in limb element length and near-full reversal (80%) of size and diameter of the ribcage and vertebrae. Aggrecan-null growth plates were devoid of matrix and lacked chondrocyte organization and differentiation, while those of the rescue embryos exhibited matrix production concomitant with partial zonation of chondrocytes having proliferative and hypertrophic morphologies. Deformation of the trachea, likely the cause of the mutation's lethality, was reduced in the rescue embryos. Aggrecan-null embryos also had abnormal patterns of COL10A1, SOX9, IHH, PTCH1, and FGFR3 mRNA expression in the growth plate. Expression of chick aggrecan in the rescue embryos notably increased COLX expression, accompanied by the reappearance of a hypertrophic zone and IHH expression. Significantly, in transgenic rescue embryos, the cell death and decreased proliferation phenotypes exhibited by the mutants were reversed; both were restored to wild-type levels. These findings suggest that aggrecan has a major role in regulating the expression of key growth factors and signaling molecules during development of cartilaginous tissue and is essential for proper chondrocyte organization, morphology, and survival during embryonic limb development.

Prevalence of amyotrophic lateral sclerosis in Texas, 1998-2003.

A prevalence study of amyotrophic lateral sclerosis (ALS) was conducted in 3 areas in Texas to enable the state health department to better respond to community concerns regarding the occurrence of ALS and to contribute to national prevalence estimates. The overall ALS point prevalence was lower than previously published US estimates. This study provides ALS prevalence estimates for Texas, including Hispanic populations.

Prevalence of psychiatric disorders in the Texas juvenile correctional system.

Most studies assessing the burden of psychiatric disorders in juvenile correctional facilities have been based on small or male-only samples or have focused on a single disorder. Using electronic data routinely collected by the Texas juvenile correctional system and its contracted medical provider organization, we estimated the prevalence of selected psychiatric disorders among youths committed to Texas juvenile correctional facilities between January 1, 2004, and December 31, 2008 (N = 11,603). Ninety-eight percent were diagnosed with at least one of the disorders. Highest estimated prevalence was for conduct disorder (83.2%), followed by any substance use disorder (75.6%), any bipolar disorder (19.4%), attention-deficit/hyperactivity disorder (18.3%), and any depressive disorder (12.6%). The estimated prevalence of psychiatric disorders among these youths was exceptionally high and showed patterns by sex, race/ethnicity, and age that were both consistent and inconsistent with other juvenile justice samples.

Astrocyte precursor response to embryonic brain injury.

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular, trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.

Eight self-administered 24-hour dietary recalls using the Internet are feasible in African Americans and Whites: the energetics study.

OBJECTIVE: To support research and to provide food and nutrition practitioners with a strong foundation for nutrient-based counseling, there is a need for affordable automated 24-hour dietary recalls. Multiple days of intake, along with repeated reports over time, are needed to achieve stable indicators of individual intakes and to support evaluation of success in meeting dietary goals because of intraindividual intake variability. Little information has been published on subject responses, participation rates, and the perceived subject burden of repeated 24-hour recalls. Our aim was to determine the willingness of subjects to conduct eight 24-hour recalls via the Internet. DESIGN: A study to validate a Web-based, automated, self-administered 24-hour recall (DietDay, Centrax Corporation, Chicago, IL). SUBJECTS/SETTING: Two-hundred and sixty-one white and African-American subjects within 50 miles of the University of California-Los Angeles participated in the study. Subjects completed 3 DietDays at the study visits and another 5 days on their own. The last 2 DietDays were completed 1 and 2 months after the final clinic visit. Subjects were notified by automatic e-mail of the need for DietDay completion, and nonresponders were followed up with personalized e-mails and phone calls. RESULTS: The perceived subject burden was minimal and, even after completing six recalls, 92% were willing to continue reporting their daily diets 1 and 2 months later. White subjects had a slightly higher rate of return, with 94% completing all eight recalls, compared to 91% of African-American subjects. Participants were able to access the Internet in their homes, offices, library, or homes of friends or family. It is also of interest that 82% of subjects believed the 24-hour recall was superior to a diet history in reflecting their normal diet. CONCLUSION: These results open up new opportunities for food and nutrition practitioners to strengthen their nutritional counseling in an efficient and affordable manner without additional time investment.

Using the web for recruitment, screen, tracking, data management, and quality control in a dietary assessment clinical validation trial.

Screening and tracking subjects and data management in clinical trials require significant investments in manpower that can be reduced through the use of web-based systems. To support a validation trial of various dietary assessment tools that required multiple clinic visits and eight repeats of online assessments, we developed an interactive web-based system to automate all levels of management of a biomarker-based clinical trial. The "Energetics System" was developed to support 1) the work of the study coordinator in recruiting, screening and tracking subject flow, 2) the need of the principal investigator to review study progress, and 3) continuous data analysis. The system was designed to automate web-based self-screening into the trial. It supported scheduling tasks and triggered tailored messaging for late and non-responders. For the investigators, it provided real-time status overviews on all subjects, created electronic case reports, supported data queries and prepared analytic data files. Encryption and multi-level password protection were used to insure data privacy. The system was programmed iteratively and required six months of a web programmer's time along with active team engagement. In this study the enhancement in speed and efficiency of recruitment and quality of data collection as a result of this system outweighed the initial investment. Web-based systems have the potential to streamline the process of recruitment and day-to-day management of clinical trials in addition to improving efficiency and quality. Because of their added value they should be considered for trials of moderate size or complexity.

Aggrecan modulation of growth plate morphogenesis.

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.

The intersection of race, poverty and risk: understanding the decision to provide services to clients and to remove children.

Studies have found that certain racial groups, particularly the children of African American families, are placed in foster care at a higher rate than children of other races. These families are also sometimes found to be afforded fewer services that might prevent these removals, relative to families of other races. It is unclear why this is so. Poverty has been suspected, and sometimes found, to be the primary cause of the disparity. Lacking in some of these analyses, however, was how risk of future abuse/neglect to the child entered into the decisions and particularly, how assumptions about race, poverty, and risk are factored into the decision-making process. It is important to understand this process if we are to find a way to correct it. The current study addresses this process.

Expanded newborn screening for biochemical disorders: the effect of a false-positive result.

BACKGROUND: Newborn screening programs now identify children with >30 biochemical genetic disorders. False-positive identifications may increase as disorders are added to screening panels. Concerns arise regarding the potential impact on parental stress, family relationships, and perceptions of the child's health. METHODS: Parents of 173 infants with false-positive screening results for a biochemical genetic disorder in the expanded newborn screening panel were compared with parents of 67 children with normal screening results. Parents completed an interview that elicited information about demographic features, child and parental health, and understanding of newborn screening. Parents also completed the parenting stress index. RESULTS: Parents in the false-positive group attained higher total scores on the PSI than did parents in the normal-screened group, scoring higher on the parent-child dysfunction subscale and the difficult child subscale. Only approximately one third of parents in the false-positive group reported knowing the correct reason for repeat screening. Mothers who reported knowing the correct reason for their child's repeat screening test experienced less total stress than did mothers who were misinformed, were not informed, or did not remember. CONCLUSIONS: False-positive screening results may affect parental stress and the parent-child relationship. Improved communication with parents regarding the need for repeat screening tests may reduce the negative impact of false-positive results.

APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region.

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.