In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form ␥-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX ⌬/⌬ ) mouse embryonic stem (ES) cells. H2AX ⌬/⌬ ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX ⌬/⌬ ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX ⌬/⌬ ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability. The DNA in eukaryotic cells is packaged into chromatin, the fundamental unit of which is the nucleosome. The nucleosome consists of DNA wrapped around an octamer of the four core histones-H2A, H2B, H3, and H4 (1). The H2A histones consist of several subfamilies that contain distinct, conserved amino-and carboxyl-terminal amino acid sequences (2). The H2AX subfamily contains a conserved carboxyl-terminal SerGln-Glu (SQE motif) amino acid sequence. This SQE motif represents the consensus in vitro phosphorylation site for members of the phosphoinositide 3-kinase related kinase (PIKK) family that includes the protein kinases DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR) (3).The repair of spontaneous and induced DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In eukaryotic cells, the two major pathways of DSB repair are nonhomologous end-joining (NHEJ) and homologous recombination (HR; refs. 4 and 5). Covalent modifications of core histones via phosphorylation, acetylation, and methylation have been proposed to form a ''histone code'' that is read by cellular proteins to facilitate downstream molecular events (6). In response to DNA damage by agents that induce DNA doublestrand breaks, Mec1, the Saccharomyces cerevisiae homologue of ATR, phosphorylates the SQE motif of H2A (7). This phosphorylation event is required for the efficient repair of chromosomal DSBs by NHEJ but does not appear to be as important for homologous recombination (7). In mammalian cells, H2AX is rapidly phosphorylated on the induction of DSBs by ionizing radiation (IR) and DNA damaging agents (8, 9), resulting in formation of ␥-H2AX foci along megabase chromatin domains flanking DNA damage sites (9).Foci of ␥-H2AX also form at the immunoglobulin heavy chain locus during class switch recombination (CSR) in activated mature B cells (10). CSR occurs between large, highly repetitive S regions and also may be initiated by DSBs (10, 11) and completed by NHEJ factors (12)(13)(14)(15). Notably, CSR is significantly impaired in the absence of H2AX (10). Earlier during lymphocyte development...

Inactivation of the XRCC4 nonhomologous end-joining factor in the mouse germ line leads to embryonic lethality, in association with apoptosis of newly generated, postmitotic neurons. We now show that conditional inactivation of the XRCC4 in nestin-expressing neuronal progenitor cells, although leading to no obvious phenotype in a WT background, leads to early onset of neuronally differentiated medulloblastomas (MBs) in a p53-deficient background. A substantial proportion of the XRCC4͞p53-deficient MBs have high-level N-myc gene amplification, often intrachromosomally in the context of complex translocations or other alterations of chromosome 12, on which N-myc resides, or extrachromosomally within double minutes. In addition, most XRCC4͞ p53-deficient MBs harbor clonal translocations of chromosome 13, which frequently involve chromosome 6 as a partner. One copy of the patched gene (Ptc), which lies on chromosome 13, was deleted in all tested XRCC4͞p53-deficient MBs in the context of translocations or interstitial deletions. In addition, Cyclin D2, a chromosome 6 gene, was amplified in a subset of tumors. Notably, amplification of Myc-family or Cyclin D2 genes and deletion of Ptc also have been observed in human MBs. We therefore conclude that, in neuronal cells of mice, the nonhomologous end-joining pathway plays a critical role in suppressing genomic instability that, in a p53-deficient background, routinely contributes to genesis of MBs with recurrent chromosomal alterations.

We have used gene-targeted mutation to assess the role of the T cell receptor delta (TCR delta) enhancer (E delta) in alphabeta and gammadelta T cell development. Mice lacking E delta exhibited no defects in alphabeta T cell development but had a severe reduction in thymic and peripheral gammadelta T cells and decreased VDJ delta rearrangements. Simultaneous deletion of both E delta and the TCR alpha enhancer (E alpha) demonstrated that residual TCR delta rearrangements were not driven by E alpha, implicating additional elements in TCR delta locus accessibility. Surprisingly, while deletion of E delta severely impaired germline TCR delta expression in double-negative thymocytes, absence of E delta did not affect expression of mature delta transcripts in gammadelta T cells. We conclude that E delta has an important role in TCR delta locus regulation at early, but not late, stages of gammadelta T cell development.

Several samples of Zinc Oxide thin films were grown using a homemade metalorganic chemical vapor deposition apparatus and tested using in-situ interferometry,photoluminescence spectrometry, ex-situ spectral reflectance, and ex-situ transmittance measurements. We found that despite having a relatively high thickness, samples grown in higher temperatures were of better crystal quality with higher exciton emission peaks, increased transmittance, and lower photoluminescence linewidth.However, kinetic energy from higher temperature growth led to increased defect-associated emission. We thus propose a model for temperature optimization in ZnO thin film growth by MOCVD wherein thermal rectification of thickness-associated defects and thermal induction of kinetic energy-induced defects must be balanced for improvements in crystal quality around the growth temperature of550·C.

Bmal1 is a core component of the molecular clock which is responsible for generating circadian rhythms in many physiological parameters, including blood pressure (BP). Sodium intake can affect circadian rhythms, as high salt consumption can increase BP and alter the expression of the molecular clock. However, the effect of low salt consumption on the circadian rhythm of BP and hormones crucial to sodium homeostasis has not been extensively studied. Using a whole-body Bmal1-knockout (KO) rat, we wanted to determine the contribution of the molecular clock to circadian BP rhythms during low salt diet (LSD, 0.0049%).We hypothesized that Bmal1-KO rats on a LSD would have lower mean BP and higher aldosterone levels in comparison to littermate controls on LSD. Male and female Bmal1-KO rats and littermate controls (WT) had telemeters implanted in the abdominal aorta at 8-10 weeks old. After a 10-day surgical recovery period, BP was continuously recorded while rats were maintained on ad libitum normal salt diet (0.49% NaCl, NSD) for 1 week. Rats were placed in metabolic cages and 12-hour urine samples collected at ZT0 and ZT12 for two days. Next, rats were placed on LSD for one week while maintaining BP measurements. This was again followed by two day, 12-hour urine collections. BP was averaged hourly and analyzed for circadian rhythmicity (cosinor). The MESOR for mean arterial pressure was significantly higher in WT (n=10, 4/10 male) and KO groups (n=8, 4/8 male) on NSD (WT: 108±2 mmHg; KO: 104±2 mmHg) compared to LSD (WT: 104±2 mmHg; KO: 102±2 mmHg; p<0.05 for diet, 2-way ANOVA). BP amplitude was significantly higher in WT rats (NSD: 7.6±0.6 mmHg; LSD: 6.9±0.7 mmHg) compared to KO rats (NSD: 5.2±0.4 mmHg; LSD: 5.3±0.9 mmHg) (p<0.05 for genotype). BP acrophase was similar between groups on both diets. Urinary aldosterone excretion (ELISA) was similar between WT and KO rats on NSD (Daytime: WT 26±5 pg/12hrs, KO 16±1 pg/12hrs; Nighttime: WT 48±8 pg/12hrs, KO 46±4 pg/12hrs) and on LSD (Daytime: WT 95±38 pg/12hrs, KO 109±44 pg/12hrs; Nighttime: WT 174±63 pg/12hrs, KO 155±47 pg/12hrs), with statistically significant overall effects of diet and time of day (p<0.05, 3-way ANOVA). Together, these data support a role for Bmal1 in control of BP amplitude in rats on NSD and LSD. This does not appear to involve changes in aldosterone since urinary excretion followed predicted patterns by time of day and salt diet, but were not impacted by loss of Bmal1 in ad libitum fed rats. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The clock gene, Bmal1, is known to regulate mitochondrial O2 consumption, but whether or how this occurs in the kidney has not been established. An estimated 95% of O2 consumption in the kidney is linked to sodium transport. We developed and used a novel Bmal1 knockout rat on a Sprague Dawley background. Male Bmal1 -/- rats do not have the typical night-day difference in Na+ excretion, while female rats maintain this pattern. We recently observed that ADP-dependent O2 consumption in permeabilized kidney tissue was significantly higher in both sexes of Bmal1 -/- rats but only during the active/dark period. We also observed a significant increase in cytochrome c oxidase activity in Bmal1 -/- rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s·mg; p=0.0031, t-test). Since these effects were observed in both male and female rats, these changes in mitochondrial activity cannot explain the difference in diurnal sodium excretion. Since mitochondrial morphology can impact oxidative phosphorylation and energy availability, we hypothesized that Bmal1 regulates mitochondrial morphology in the kidney. Male and female global Bmal1 +/+ and Bmal1 -/- rats at 12-14 weeks of age were maintained in regular 12:12 LD cycles. Outer renal medullary tissue was dissected and prepared for mRNA expression and transmission electron microscopy (EM). For EM, tissues were obtained at light (ZT2-4) or dark (ZT14-16) periods to correspond with the minimum and maximum whole-body energy consumption and peak and trough Bmal1 protein expression. Digital droplet PCR mRNA expression was conducted from tissue collected at 4hr intervals over 24hrs. EM images appear to show an elongation of mitochondrial morphology in females but not male Bmal1 -/- during the active time of day. Mitofusion1 (Mfn1) and fission 1 (Fis1) are genes that regulate mitochondrial structural dynamics. Mfn1 and Fis1 mRNA expression over 24hrs showed a significant interaction between genotype and time of day in male rats (p=0.004 and p=0.020, respectively, 2way ANOVA). In contrast, there were no significant differences in overall Mfn1 or Fis1 expression between female Bmal1 +/+ and Bmal1 -/- rats. Our findings demonstrate that Bmal1 is crucial in maintaining mitochondrial respiration coupling and ATP generation in the kidney. Furthermore, we suggest that female Bmal1 -/- rats can better compensate for impaired respiration by mitochondrial morphological changes when the molecular clock is dysfunctional. We further propose that the rhythmicity of Na+ excretion in females, but not male, Bmal1 -/- rats is due to adjustments in mitochondrial morphology. T32 HL007457, P01HL136267, AHA 908953 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.