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.

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.

Modeling Cellular Noise Underlying Heterogeneous Cell Responses in the Epidermal Growth Factor Signaling Pathway.

Cellular heterogeneity, which plays an essential role in biological phenomena, such as drug resistance and migration, is considered to arise from intrinsic (i.e., reaction kinetics) and extrinsic (i.e., protein variability) noise in the cell. However, the mechanistic effects of these types of noise to determine the heterogeneity of signal responses have not been elucidated. Here, we report that the output of epidermal growth factor (EGF) signaling activity is modulated by cellular noise, particularly by extrinsic noise of particular signaling components in the pathway. We developed a mathematical model of the EGF signaling pathway incorporating regulation between extracellular signal-regulated kinase (ERK) and nuclear pore complex (NPC), which is necessary for switch-like activation of the nuclear ERK response. As the threshold of switch-like behavior is more sensitive to perturbations than the graded response, the effect of biological noise is potentially critical for cell fate decision. Our simulation analysis indicated that extrinsic noise, but not intrinsic noise, contributes to cell-to-cell heterogeneity of nuclear ERK. In addition, we accurately estimated variations in abundance of the signal proteins between individual cells by direct comparison of experimental data with simulation results using Apparent Measurement Error (AME). AME was constant regardless of whether the protein levels varied in a correlated manner, while covariation among proteins influenced cell-to-cell heterogeneity of nuclear ERK, suppressing the variation. Simulations using the estimated protein abundances showed that each protein species has different effects on cell-to-cell variation in the nuclear ERK response. In particular, variability of EGF receptor, Ras, Raf, and MEK strongly influenced cellular heterogeneity, while others did not. Overall, our results indicated that cellular heterogeneity in response to EGF is strongly driven by extrinsic noise, and that such heterogeneity results from variability of particular protein species that function as sensitive nodes, which may contribute to the pathogenesis of human diseases.

A Dictyostelium cellobiohydrolase orthologue that affects developmental timing.

Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound - tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug - late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2-4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc (oe) strain displayed 4-methylumbelliferyl ß-D-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.

[Case of neuroleptic malignant syndrome following open heart surgery for thoracic aortic aneurysm with parkinson's disease].

An 80-year-old woman with Parkinson's disease was scheduled for open heart surgery to repair thoracic aortic aneurysm. Parkinson's symptoms were normally treated using oral levodopa (200 mg), selegiline-hydrochloride (5 mg), bromocriptine-mesilate (2 mg), and amantadine-hydrochloride (200 mg) daily. On the day before surgery, levodopa 50mg was infused intravenously. Another 25 mg of levodopa was infused immediately after surgery. Twenty hours later, the patient developed tremors, heyperventilation, but no obvious muscle rigidity. Two days after surgery, the patient exhibited high fever, hydropoiesis, elevated creatine kinase, and a rise in blood leukocytes. She was diagnosed with neuroleptic malignant syndrome. She was intubated, and received dantrolene sodium. Symptoms of neuroleptic malignant syndrome disappeared on the fourth postoperative day. The stress of open heart surgery, specifically extracorporeal circulation and concomitant dilution of levodopa, triggered neuroleptic malignant syndrome in this patient. Parkinson's patients require higher doses of levodopa prior to surgery to compensate and prevent neuroleptic malignant syndrome after surgery.

[Effect on fetal umbilical arterial blood of administration of vasopressors for hypotension after spinal anesthesia during cesarean section].

Spinal anesthesia was performed in 40 patients undergoing cesarean section. When systolic blood pressure dropped below 100 mmHg, phenylephrine 100-200 microg (P group) or ephedrine 5-10 mg (E group) was administered. The pH of the umbilical arterial blood was collected after delivery of the baby. Apgar scores, and maternal systolic blood pressure and heart rate before and after each drug administration were compared retrospectively. The umbilical arterial pH and Apgar scores tended to be slightly higher in the P group, but there was no significant difference between the two groups. The rate of blood pressure elevation was 27% in the P group and 41% in the E group. The heart rate decreased significantly in the P group. There was no significant difference in the systolic blood pressure before administration of each drug. Recently, it is reported that the umbilical arterial pH is higher in cases in which phenylephrine is used for hypotension after spinal anesthesia during a cesarean section. However, the optimal dose of phenylephrine is debatable and has not been established. More studies are necessary to determine which drugs should be selected according to the maternal condition.

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.

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.

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.

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.

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.

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.

Inferring a nonlinear biochemical network model from a heterogeneous single-cell time course data.

Mathematical modeling and analysis of biochemical reaction networks are key routines in computational systems biology and biophysics; however, it remains difficult to choose the most valid model. Here, we propose a computational framework for data-driven and systematic inference of a nonlinear biochemical network model. The framework is based on the expectation-maximization algorithm combined with particle smoother and sparse regularization techniques. In this method, a "redundant" model consisting of an excessive number of nodes and regulatory paths is iteratively updated by eliminating unnecessary paths, resulting in an inference of the most likely model. Using artificial single-cell time-course data showing heterogeneous oscillatory behaviors, we demonstrated that this algorithm successfully inferred the true network without any prior knowledge of network topology or parameter values. Furthermore, we showed that both the regulatory paths among nodes and the optimal number of nodes in the network could be systematically determined. The method presented in this study provides a general framework for inferring a nonlinear biochemical network model from heterogeneous single-cell time-course data.

Massive ameloblastoma: A case report of difficult fiberoptic intubation.

INTRODUCTION: Intubation can sometimes be difficult in patients with lesions in the mouth floor. Ameloblastoma is a frequently encountered tumor of the maxillofacial area. An extensive lesion might occupy the floor of the mouth, prevent displacement of the tongue, limiting the space for inserting a laryngoscope blade and resulting in difficult intubation even with fiberoptic bronchoscopy. CASE PRESENTATION: A 66-year-old man (67 kg; 171 cm) with a mental swelling was diagnosed with ameloblastoma and scheduled for surgical resection. The tumor was extensive, occupying most of the anterior floor of the mouth. We were concerned about impossible direct laryngoscopy because the massive tumor in the floor of the mouth compressed the base of the tongue against the posterior wall of the pharynx, restricting the space for inserting the laryngoscope blade. Therefore, we planned to perform awake nasal fiberoptic intubation to secure the airway. Although the procedure was complicated by the massive tumor, successful intubation was achieved by hand-assisted alteration of the direction of the endotracheal tube (ETT) under direct laryngoscopy. DISCUSSION: Awake fiberoptic intubation was complicated by the tumor protrusion to deviate the ETT. Discovering of the ETT deviation by the insufficient blade insertion facilitated visualizing the vocal cords with the fiberoptic scope. CONCLUSION: Identification of ETT deviation even with insufficient blade insertion and hand-assisted alteration of the direction of the ETT might raise the chances of successful fiberoptic intubation. The anesthesiologist should be aware of the likelihood of failed fiberoptic intubation and plan for alternative approaches to secure the airway.

Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling.

The phosphorylation cascade in the extracellular signal-regulated kinase (ERK) pathway is a versatile reaction network motif that can potentially act as a switch, oscillator or memory. Nevertheless, there is accumulating evidence that the phosphorylation response is mostly linear to extracellular signals in mammalian cells. Here we find that subsequent nuclear translocation gives rise to a switch-like increase in nuclear ERK concentration in response to signal input. The switch-like response disappears in the presence of ERK inhibitor, suggesting the existence of autoregulatory mechanisms for ERK nuclear translocation involved in conversion from a graded to a switch-like response. In vitro reconstruction of ERK nuclear translocation indicates that ERK-mediated phosphorylation of nucleoporins regulates ERK translocation. A mathematical model and knockdown experiments suggest a contribution of nucleoporins to regulation of the ERK nuclear translocation response. Taken together, this study provides evidence that nuclear translocation with autoregulatory mechanisms acts as a switch in ERK signalling.

Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary.

Cell polarity arises through the spatial segregation of polarity regulators. PAR proteins are polarity regulators that localize asymmetrically to two opposing cortical domains. However, it is unclear how the spatially segregated PAR proteins interact to maintain their mutually exclusive partitioning. Here, single-molecule detection analysis in Caenorhabditis elegans embryos reveals that cortical PAR-2 diffuses only short distances, and, as a result, most PAR-2 molecules associate and dissociate from the cortex without crossing into the opposing domain. Our results show that cortical PAR-2 asymmetry is maintained by the local exchange reactions that occur at the cortical-cytoplasmic boundary. Additionally, we demonstrate that local exchange reactions are sufficient to maintain cortical asymmetry in a parameter-free mathematical model. These findings suggest that anterior and posterior PAR proteins primarily interact through the cytoplasmic pool and not via cortical diffusion.

A Computational Framework for Bioimaging Simulation.

Using bioimaging technology, biologists have attempted to identify and document analytical interpretations that underlie biological phenomena in biological cells. Theoretical biology aims at distilling those interpretations into knowledge in the mathematical form of biochemical reaction networks and understanding how higher level functions emerge from the combined action of biomolecules. However, there still remain formidable challenges in bridging the gap between bioimaging and mathematical modeling. Generally, measurements using fluorescence microscopy systems are influenced by systematic effects that arise from stochastic nature of biological cells, the imaging apparatus, and optical physics. Such systematic effects are always present in all bioimaging systems and hinder quantitative comparison between the cell model and bioimages. Computational tools for such a comparison are still unavailable. Thus, in this work, we present a computational framework for handling the parameters of the cell models and the optical physics governing bioimaging systems. Simulation using this framework can generate digital images of cell simulation results after accounting for the systematic effects. We then demonstrate that such a framework enables comparison at the level of photon-counting units.

Computational analysis of associations between alternative splicing and histone modifications.

Pre-mRNA splicing is a complex process involving combinatorial effects of cis- and trans-elements. Here, we focused on histone modifications as typical trans-regulatory elements and performed systematic analyses of associations between splicing patterns and histone modifications by using publicly available ChIP-Seq, mRNA-Seq, and exon-array data obtained in two human cell lines. We found that several types of histone modifications including H3K36me3 were associated with the inclusion or exclusion of alternative exons. Furthermore, we observed that the levels of H3K36me3 and H3K79me1 in the cell lines were well correlated with the differences in alternative splicing patterns between the cell lines.