Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation.

Early embryos often have relatively unstructured chromatin that lacks active and inactive domains typical of differentiated cells. In many species, these regulatory domains are established during zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 fast, reductive, syncytial nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. These divisions incorporate maternally-loaded, cytoplasmic pools of histones into chromatin. Previous work found that chromatin incorporation of replication-coupled histone H3 decreases while its variant H3.3 increases in the cell cycles leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription as well as heterochromatin, suggesting a link between H3.3 incorporation and ZGA. Here, we examine the factors that contribute to H3.3 incorporation at ZGA. We identify a more rapid decrease in the nuclear availability of H3 than H3.3 over the final pre-ZGA cycles. We also observe an N/C ratio-dependent increase in H3.3 incorporation in mutant embryos with non-uniform local N/C ratios. We find that chaperone binding, not gene expression, controls incorporation patterns using H3/H3.3 chimeric proteins at the endogenous H3.3A locus. We test the specificity of the H3.3 chaperone pathways for H3.3 incorporation using Hira (H3.3 chaperone) mutant embryos. Overall, we propose a model in which local N/C ratios and specific chaperone binding regulate differential incorporation of H3.3 during ZGA.

A parasympathetic neurotransmitter induces myoepithelial cell differentiation during salivary gland development.

Myoepithelial cells (MECs) are responsible for receiving stimuli from the central nervous system and translating their responses into the form of secretion into glandular tissue, including salivary glands (SG), sweet glands, and mammary glands. SG MECs cause the secretion of serous saliva by contracting of acini/ductal cells with acetylcholine (Ach) from parasympathetic nerves via muscarinic receptors. To response the parasympathetic physiological stimulation, SG epithelial cell-derived MECs are supposed to be induced and placed adjacent to parasympathetic system nerve ends in SGs by forming a neuro-myoepithelial junction. For salivary secretion to function under parasympathetic control, therefore, specific regions of salivary gland epithelial cells must be mapped and the epithelium near the nerve must differentiate into MECs in order to form a nerve-myoepithelial junction during organogenesis. We hypothesized that the epithelium near the parasympathetic nerves is induced the differentiation into MECs by which the neurotransmitter acetylcholine via muscarinic receptors. qPCR and whole-mount immunohistochemical analysis in ex vivo organ culture system revealed that SG epithelial cells near a parasympathetic nerve were found to be induced to differentiate into MECs via the cholinergic receptor muscarinic 1 by carbachol (CCh), an acetylcholine agonist. In addition, CCh stimulated ERK and Akt signaling for the induction of MEC differentiation in rat submandibular gland epithelial cells. These findings indicate that muscarinic action is required for the induction of MECs and formation of a neuro-myoepithelial junction in developing SGs. This study proposes a novel concept for tissue architecture to form a neuro-myoepithelial junction during neurofunctional organogenesis including SGs.

Versatile roles for histones in early development.

The nuclear environment changes dramatically over the course of early development. Histones are core chromatin components that play critical roles in regulating gene expression and nuclear architecture. Additionally, the embryos of many species, including Drosophila, Zebrafish, and Xenopus use the availability of maternally deposited histones to time critical early embryonic events including cell cycle slowing and zygotic genome activation. Here, we review recent insights into how histones control early development. We first discuss the regulation of chromatin functions through interaction of histones and transcription factors, incorporation of variant histones, and histone post-translational modifications. We also highlight emerging roles for histones as developmental regulators independent of chromatin association.

Modeling the role for nuclear import dynamics in the early embryonic cell cycle.

Nuclear composition determines nuclear function. The early embryos of many species begin life with large pools of maternally provided components that become rapidly imported into an increasing number of nuclei as the cells undergo repeated cleavage divisions. Because early cell cycles are too fast for nuclei to achieve steady-state nucleocytoplasmic partitioning, the composition of cleavage stage nuclei is likely dominated by nuclear import. The end of the rapid cleavage stage and onset of major zygotic transcription, known as the mid-blastula transition (MBT), is controlled by the ratio of nuclei/cytoplasm, indicating that changes in nuclear composition likely mediate MBT timing. Here, we explore how different nuclear import regimes can affect protein accumulation in the nucleus in the early Drosophila embryo. We find that nuclear import differs dramatically for a general nuclear cargo (NLS (nuclear localization signal)-mRFP) and a proposed MBT regulator (histone H3). We show that nuclear import rates of NLS-mRFP in a given nucleus remain relatively unchanged throughout the cleavage cycles, whereas those of H3 halve with each cycle. We model these two distinct modes of nuclear import as "nucleus-limited" and "import-limited" and examine how the two different modes can contribute to different protein accumulation dynamics. Finally, we incorporate these distinct modes of nuclear import into a model for cell-cycle regulation at the MBT and find that the import-limited H3 dynamics contribute to increased robustness and allow for stepwise cell-cycle slowing at the MBT.

Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo.

The DNA damage checkpoint is crucial to protect genome integrity.1,2 However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio)3-7 and the activation of the checkpoint kinase, Chk1.8-17 How Chk1 activity is coupled to the N/C ratio has remained poorly understood. Here, we show that dynamic changes in histone H3 availability in response to the increasing N/C ratio control Chk1 activity and thus time the MBT in the Drosophila embryo. We show that excess H3 in the early cycles interferes with cell-cycle slowing independent of chromatin incorporation. We find that the N-terminal tail of H3 acts as a competitive inhibitor of Chk1 in vitro and reduces Chk1 activity in vivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, we show that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression.

Regulation of miR-1-Mediated Connexin 43 Expression and Cell Proliferation in Dental Epithelial Cells.

Many genes encoding growth factors, receptors, and transcription factors are induced by the epithelial-mesenchymal interaction during tooth development. Recently, numerous functions of microRNAs (miRNAs) are reportedly involved in organogenesis and disease. miRNAs regulate gene expression by inhibiting translation and destabilizing mRNAs. However, the expression and function of miRNAs in tooth development remain poorly understood. This study aimed to analyze the expression of miRNAs produced during tooth development using a microarray system to clarify the role of miRNAs in dental development. miR-1 showed a unique expression pattern in the developing tooth. miR-1 expression in the tooth germ peaked on embryonic day 16.5, decreasing gradually on postnatal days 1 and 3. An in situ hybridization assay revealed that miR-1 is expressed at the cervical loop of the dental epithelium. The expression of miR-1 and connexin (Cx) 43, a target of miR-1, were inversely correlated both in vitro and in vivo. Knockdown of miR-1 induced the expression of Cx43 in dental epithelial cells. Interestingly, cells with miR-1 downregulation proliferated slower than the control cells. Immunocytochemistry revealed that Cx43 in cells with miR-1 knockdown formed both cell-cell gap junctions and hemichannels at the plasma membrane. Furthermore, the rate of ATP release was higher in cells with miR-1 knockdown than in control cells. Furthermore, Cx43 downregulation in developing molars was observed in Epiprofin-knockout mice, along with the induction of miR-1 expression. These results suggest that the expression pattern of Cx43 is modulated by miR-1 to control cell proliferation activity during dental epithelial cell differentiation.

Effects of low-dose ketamine infusion on remifentanil-induced acute opioid tolerance and the inflammatory response in patients undergoing orthognathic surgery.

PURPOSE: Remifentanil is associated with acute opioid tolerance that can lead to increased postoperative consumption of opioid analgesics. The purpose of this study was to determine whether a low dose of ketamine prevents remifentanil-induced acute opioid tolerance and affects the neutrophil-lymphocyte ratio (NLR), a newly recognized biomarker of inflammation. MATERIALS AND METHODS: Forty patients undergoing orthognathic surgery were enrolled in this prospective, randomized, double-blind study and randomly assigned to intraoperative administration of one of the following anesthetic regimens: high-dose remifentanil (0.6 µg/kg/minute); low-dose remifentanil (0.2 µg/kg/minute); or high-dose remifentanil with ketamine (remifentanil 0.6 µg/kg/minute with 0.5 mg/kg ketamine just after induction followed by an intraoperative infusion of ketamine 5 µg/kg/minute until wound closure). Fentanyl by intravenous patient-controlled analgesia was used for postoperative pain control. Visual Analog Scale pain scores and fentanyl consumption were recorded in the first 24 hours postoperatively. Perioperative serum C-reactive protein level and NLR were also determined. RESULTS: Baseline characteristics were similar in the three study groups. There were no between-group differences in Visual Analog Scale pain scores during the study period. The high-dose remifentanil group had a significantly higher requirement for fentanyl than the other two groups. Addition of ketamine did not affect the C-reactive protein level but increased the NLR; this increase was associated with decreased fentanyl consumption. CONCLUSION: High-dose intraoperative remifentanil induced postoperative acute opioid tolerance that was prevented by infusion of low-dose ketamine. Ketamine increased the postoperative NLR associated with decreased fentanyl requirement for postoperative pain control.

Dynamics of Free and Chromatin-Bound Histone H3 during Early Embryogenesis.

During zygotic genome activation (ZGA), the chromatin environment undergoes profound changes, including the formation of topologically associated domains, refinements in nucleosome positioning on promoters, and the emergence of heterochromatin [1-4]. In many organisms, including Drosophila, ZGA is associated with the end of a period of extremely rapid, exponential cleavage divisions that are facilitated by large maternally provided pools of nuclear components. It is therefore imperative that we understand how the supply of chromatin components relative to the exponentially increasing demand affects nuclear and chromatin composition during early embryogenesis. Here, we examine the nuclear trafficking and chromatin dynamics of histones during the cleavage divisions in Drosophila using a photo-switchable H3-Dendra2 reporter. We observe that total H3-Dendra2 in the nucleus decreases with each cleavage cycle. This change in nuclear composition is due to depletion of large pools (>50%) of free protein that are present in the early cycles. We find that the per nucleus import rate halves with each cycle and construct a mathematical model in which increasing histone demand determines the dynamics of nuclear H3 supply. Finally, we show that these changes in H3 availability correspond to a large (∼40%) reduction in global H3 occupancy on the chromatin, which is compensated by the increased incorporation of H3.3. The observed changes in free nuclear H3 and chromatin composition may contribute to the cell-cycle slowing, changes in chromatin structure, and the onset of transcription associated with this developmental stage.

Acute Management of Massive Epistaxis After Nasotracheal Extubation.

Epistaxis is one of the most common complications of nasotracheal intubation and can be life-threatening. However, there is little discussion in the current literature on the acute management of massive epistaxis after nasotracheal extubation. This is a report of 2 patients who experienced severe unanticipated nasal bleeding immediately after extubation, 1 after a surgical procedure for oral cancer and another after restorative dental treatment. In both cases the significant epistaxis was managed successfully with a Foley balloon catheter used to pack the posterior nasal cavity. The Foley catheter technique may be useful for managing and arresting sudden postextubation epistaxis.

Expression of ß-endorphin in peripheral tissues after systemic administration of lipopolysaccharide as a model of endotoxic shock in mice.

BACKGROUND: Endogenous ß-endorphin is delivered exclusively from the pituitary gland in various stressful conditions and plays an essential role in the nervous system. Recently, a few studies demonstrated peripheral endogenous opioid secretion from immune cells at inflammatory sites. Here, we investigated the expression of ß-endorphin, the most powerful endogenous opioid peptide, in peripheral tissues in response to systemic administration of lipopolysaccharide in mice. METHODS: Male C57BL/6N mice received intravenously administered lipopolysaccharide to induce an endotoxic shock-like condition. mRNA for proopiomelanocortin, a precursor of ß-endorphin, was quantified in peripheral blood cells, liver and spleen. ß-endorphin peptide was measured in the liver and spleen. RESULTS: Expression of proopiomelanocortin mRNA was detected in peripheral tissues after systemic administration of lipopolysaccharide. Lipopolysaccharide also induced ß-endorphin expression in the liver and spleen. CONCLUSION: Expression of proopiomelanocortin mRNA and ß-endorphin was detected in peripheral tissues after systemic administration of lipopolysaccharide. These results provide new evidence that peripheral endogenous opioids can be produced not only as a result of local inflammation but also by severe systemic stress such as endotoxic shock. Further study is required to clarify the role of peripheral ß-endorphin during endotoxic shock.