Deficiencies in the DNA Binding Protein ARID3a Alter Chromatin Structures Important for Early Human Erythropoiesis.

ARID3a is a DNA-binding protein important for normal hematopoiesis in mice and for in vitro lymphocyte development in human cultures. ARID3a knockout mice die in utero with defects in both early hematopoietic stem cell populations and erythropoiesis. Recent transcriptome analyses in human erythropoietic systems revealed increases in ARID3a transcripts implicating potential roles for ARID3a in human erythrocyte development. However, ARID3a transcript levels do not faithfully reflect protein levels in many cells, and the functions and requirements for ARID3a protein in those systems have not been explored. We used the erythroleukemic cell line K562 as a model to elucidate functions of ARID3a protein in early human erythropoiesis. ARID3a knockdown of hemin-stimulated K562 cells resulted in lack of fetal globin production and modifications in gene expression. Temporal RNA sequencing data link ARID3a expression with the important erythroid regulators Gata1, Gata2, and Klf1 Ablation of ARID3a using CRISPR-Cas9 further demonstrated it is required to maintain chromatin structures associated with erythropoietic differentiation potential. These data demonstrate that the ARID3a protein is required for early erythropoietic events and provide evidence for the requirement of ARID3a functions for proper maintenance of appropriate chromatin structures.

Comparison of Antimicrobial Efficacy of Diode Laser, Ultrasonic Activated and Conventional Irrigation with 2.5% NaOCl during RCT: An Interventional Study.

AIM AND OBJECTIVE: The aim and objective of the study was to evaluate and compare the antimicrobial efficacy of a diode laser irradiation, ultrasonic activated and conventional irrigation with 2.5% NaOCl on obligatory and facultative anaerobic bacteria in single-rooted canals. MATERIALS AND METHODS: Total of 60 permanent maxillary and mandibular single-rooted (single canal) anterior teeth were selected. First microbial sample (S1) was collected after access opening and working length determination, using a sterile paper point. Cleaning and shaping were performed, with each instrument change accompanied by irrigation using 2 mL 2.5% NaOCl. After cleaning and shaping, disinfection protocol using diode laser (group1), ultrasonic activated irrigation with 2.5% NaOCl (group 2) and conventional irrigation with 2.5% NaOCl (group 3) was performed and second microbial sample (S2) was obtained. The colony characters of each type of growth on each media were noted and the organisms were identified using standard biochemical reactions. RESULT: Gram-positive and gram-negative facultative anaerobe were predominantly isolated from the culture, and the highest reduction of the microbial count was seen in diode laser group with 60.92% followed by the ultrasonic group with 47.22% reduction and least reduction was observed in conventional irrigation with the ultrasonic group with 37.97%. The results were statistically significant with p-value <0.05. CONCLUSION: Diode laser disinfection showed the highest reduction of microbial count compared to ultrasonic activated and conventional needle irrigation with 2.5% NaOCl group. CLINICAL SIGNIFICANCE: This study will help us to choose wisely between various irrigating methods and protocols. Diode laser in our study has shown superior disinfection of the root canals compared to others.

TLR engagement induces ARID3a in human blood hematopoietic progenitors and modulates IFNα production.

The DNA binding protein AT-rich interacting domain 3a (ARID3a)2 is expressed in healthy human hematopoietic cord blood progenitors where its modulation influences myeloid versus B lineage development. ARID3a is also variably expressed in subsets of adult peripheral blood hematopoietic progenitors where the consequences of ARID3a expression are unknown. In B lymphocytes, Toll-like receptor (TLR)3 signaling induces ARID3a expression in association with Type I interferon inflammatory cytokines. We hypothesized that TLR ligand stimulation of peripheral blood hematopoietic progenitors would induce ARID3a expression resulting in interferon production, and potentially influencing lineage decisions. Our data revealed that the TLR9 agonist CpG induces ARID3a expression with interferon alpha synthesis in human hematopoietic progenitors. However, ARID3a expression was not associated with increased B lineage development. These results demonstrate the need for further experiments to better define how pathogen-associated responses influence hematopoiesis.

Transient Cerebral Ischemia Promotes Brain Mitochondrial Dysfunction and Exacerbates Cognitive Impairments in Young 5xFAD Mice.

Alzheimer's disease (AD) is heterogeneous and multifactorial neurological disorder; and the risk factors of AD still remain elusive. Recent studies have highlighted the role of vascular factors in promoting the progression of AD and have suggested that ischemic events increase the incidence of AD. However, the detailed mechanisms linking ischemic insult to the progression of AD is still largely undetermined. In this study, we have established a transient cerebral ischemia model on young 5xFAD mice and their non-transgenic (nonTg) littermates by the transient occlusion of bilateral common carotid arteries. We have found that transient cerebral ischemia significantly exacerbates brain mitochondrial dysfunction including mitochondrial respiration deficits, oxidative stress as well as suppressed levels of mitochondrial fusion proteins including optic atrophy 1 (OPA1) and mitofusin 2 (MFN2) in young 5xFAD mice resulting in aggravated spatial learning and memory. Intriguingly, transient cerebral ischemia did not induce elevation in the levels of cortical or mitochondrial Amyloid beta (Aß)1-40 or 1-42 levels in 5xFAD mice. In addition, the glucose- and oxygen-deprivation-induced apoptotic neuronal death in Aß-treated neurons was significantly mitigated by mitochondria-targeted antioxidant mitotempo which suppresses mitochondrial superoxide levels. Therefore, the simplest interpretation of our results is that young 5xFAD mice with pre-existing AD-like mitochondrial dysfunction are more susceptible to the effects of transient cerebral ischemia; and ischemic events may exacerbate dementia and worsen the outcome of AD patients by exacerbating mitochondrial dysfunction.

Early detection of subclinical visual damage after blast-mediated TBI enables prevention of chronic visual deficit by treatment with P7C3-S243.

PURPOSE: Traumatic brain injury (TBI) frequently leads to chronic visual dysfunction. The purpose of this study was to investigate the effect of TBI on retinal ganglion cells (RGCs), and to test whether treatment with the novel neuroprotective compound P7C3-S243 could prevent in vivo functional deficits in the visual system. METHODS: Blast-mediated TBI was modeled using an enclosed over-pressure blast chamber. The RGC physiology was evaluated using a multielectrode array and pattern electroretinogram (PERG). Histological analysis of RGC dendritic field and cell number were evaluated at the end of the study. Visual outcome measures also were evaluated based on treatment of mice with P7C3-S243 or vehicle control. RESULTS: We show that deficits in neutral position PERG after blast-mediated TBI occur in a temporally bimodal fashion, with temporary recovery 4 weeks after injury followed by chronically persistent dysfunction 12 weeks later. This later time point is associated with development of dendritic abnormalities and irreversible death of RGCs. We also demonstrate that ongoing pathologic processes during the temporary recovery latent period (including abnormalities of RGC physiology) lead to future dysfunction of the visual system. We report that modification of PERG to provocative postural tilt testing elicits changes in PERG measurements that correlate with a key in vitro measures of damage: the spontaneous and light-evoked activity of RGCs. Treatment with P7C3-S243 immediately after injury and throughout the temporary recovery latent period protects mice from developing chronic visual system dysfunction. CONCLUSIONS: Provocative PERG testing serves as a noninvasive test in the living organism to identify early damage to the visual system, which may reflect corresponding damage in the brain that is not otherwise detectable by noninvasive means. This provides the basis for developing an earlier diagnostic test to identify patients at risk for developing chronic CNS and visual system damage after TBI at an earlier stage when treatments may be more effective in preventing these sequelae. In addition, treatment with the neuroprotective agent P7C3-S243 after TBI protects from visual system dysfunction after TBI.

Serum amyloid A protects murine macrophages from lethal toxin-mediated death.

Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.

Developmental time course distinguishes changes in spontaneous and light-evoked retinal ganglion cell activity in rd1 and rd10 mice.

In a subset of hereditary retinal diseases, early photoreceptor degeneration causes rapidly progressive blindness in children. To better understand how retinal development may interact with degenerative processes, we compared spontaneous and light-evoked activity among retinal ganglion cells in rd1 and rd10 mice, strains with closely related retinal disease. In each, a mutation in the Pde6b gene causes photoreceptor dysfunction and death, but in rd10 mice degeneration starts after a peak in developmental plasticity of retinal circuitry and thereafter progresses more slowly. In vitro multielectrode action potential recordings revealed that spontaneous waves of correlated ganglion cell activity comparable to those in wild-type mice were present in rd1 and rd10 retinas before eye opening [postnatal day (P) 7 to P8]. In both strains, spontaneous firing rates increased by P14-P15 and were many times higher by 4-6 wk of age. Among rd1 ganglion cells, all responses to light had disappeared by ~P28, yet in rd10 retinas vigorous ON and OFF responses were maintained well beyond this age and were not completely lost until after P60. This difference in developmental time course separates mechanisms underlying the hyperactivity from those that alter light-driven responses in rd10 retinas. Moreover, several broad physiological groups of cells remained identifiable according to response polarity and time course as late as P60. This raises hope that visual function might be preserved or restored despite ganglion cell hyperactivity seen in inherited retinal degenerations, particularly if treatment or manipulation of early developmental plasticity were to be timed appropriately.

Anti-nuclear antibody production and autoimmunity in transgenic mice that overexpress the transcription factor Bright.

The B cell-restricted transcription factor, B cell regulator of Ig(H) transcription (Bright), up-regulates Ig H chain transcription 3- to 7-fold in activated B cells in vitro. Bright function is dependent upon both active Bruton's tyrosine kinase and its substrate, the transcription factor, TFII-I. In mouse and human B lymphocytes, Bright transcription is down-regulated in mature B cells, and its expression is tightly regulated during B cell differentiation. To determine how Bright expression affects B cell development, transgenic mice were generated that express Bright constitutively in all B lineage cells. These mice exhibited increases in total B220(+) B lymphocyte lineage cells in the bone marrow, but the relative percentages of the individual subpopulations were not altered. Splenic immature transitional B cells were significantly expanded both in total cell numbers and as increased percentages of cells relative to other B cell subpopulations. Serum Ig levels, particularly IgG isotypes, were increased slightly in the Bright-transgenic mice compared with littermate controls. However, immunization studies suggest that responses to all foreign Ags were not increased globally. Moreover, 4-wk-old Bright-transgenic mice produced anti-nuclear Abs. Older animals developed Ab deposits in the kidney glomeruli, but did not succumb to further autoimmune sequelae. These data indicate that enhanced Bright expression results in failure to maintain B cell tolerance and suggest a previously unappreciated role for Bright regulation in immature B cells. Bright is the first B cell-restricted transcription factor demonstrated to induce autoimmunity. Therefore, the Bright transgenics provide a novel model system for future analyses of B cell autoreactivity.

Development, maintenance, and function of the adrenal gland in early postnatal proopiomelanocortin-null mutant mice.

Adult mouse mutants homozygous for an engineered proopiomelanocortin (POMC)-null allele lack macroscopically distinct adrenal glands and circulating adrenal hormones. To understand the basis for this adrenal defect, we compared the development of adrenal primordia in POMC-null mice and littermate controls. POMC-null mutant mice are born with adrenal glands that are morphologically indistinguishable from those of their wild-type littermates. However, in mutants adrenal cells fail to proliferate postnatally and adrenals atrophy until they have disappeared macroscopically in the adult. While present, mutant adrenals are differentiated as evidenced by the presence of enzymes for the final steps in the synthesis of corticosterone, aldosterone, and catecholamines. However, in contrast to adrenals of wild-type littermates, adrenals of POMC-null mutants do not produce corticosterone, not even in response to acute stimulation with exogenous ACTH. They do produce aldosterone; however, it is produced at reduced levels correlating with adrenal size. Transplantation of POMC-null mutant adrenals to adrenalectomized wild-type littermates results in adrenals with normal morphology and production of both corticosterone and aldosterone. These findings demonstrate that POMC peptides are not required for prenatal adrenal development and that POMC peptides in addition to ACTH are required for postnatal proliferation and maintenance of adrenal structures capable of producing both glucocorticoids and mineralocorticoids.