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Summary: This article is concerned with the finite‐time adaptive event‐triggered control for stochastic nonstrict‐feedback nonlinear systems, which are under asymmetric full‐state time‐varying constraints. By utilizing the hyperbolic tangent function as a nonlinear mapping approach, the system with time‐varying constraints is transformed into an unconstrained stochastic nonlinear system. To reduce the communication burden and energy consumption, adaptive event‐triggered control (ETC) is extended to the converted stochastic nonstrict‐feedback system combining the dynamic surface control (DSC) technology and the characteristic of Gaussian function. The designed controller can make the system semi‐globally finite‐time stable in probability (SGFSP), which ensures a fast convergence speed. Moreover, to approximate the unknown nonlinear functions, radial basis function neural networks (RBF NNs) are adopted. The proposed control scheme can be confirmed in the fact that all signals in the closed‐loop system are semi‐globally uniformly bounded in probability, and the tracking error converges rapidly to a small neighborhood of zero in finite time. Two simulation results are given to demonstrate the effectiveness of the proposed controller. [ABSTRACT FROM AUTHOR]

Abstract: We examined the role and molecular mechanism of cADPR action on Ca2+ spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1±0.87 currents/min from 1.2±0.36 currents/min (control) and to 179.8±48.7pA from 36.4±22.6pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca2+ spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca2+ spark frequency was increased by about 3.4-fold, peak Ca2+ (F/F0) increased to 1.72±0.57 from 1.56±0.13, size increased to 2.86±0.26μM from 1.92±0.14μM, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca2+ spark properties, and cADPR increased F/F0 without affecting Ca2+ spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca2+ load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca2+ release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle. [Copyright &y& Elsevier]

Abstract: Non-coding exons of ε-globin mRNA originating within the 236kb upstream region of the ε-globin gene were identified in human primary tissues and K562 cells. One predominant type of upstream ε mRNA, which originated in the −76kb region 5′ to the ε gene, was present in human primary tissues, whereas 11 other isoforms were identified in K562 cells. Fragment from the −76kb region possessed promoter activity and a prominent DNase I hypersensitive site was formed in the region ∼2kb 5′ to the −76kb promoter in human fetal liver, but not in K562 cells. The promoter activity in the −236kb region resided in a retrotransposon in K562 cells. A DNase I hypersensitive site was formed at the −236kb promoter in K562 cells, but not in human fetal liver. We discussed these results in the context of intergenic transcription and chromatin opening in the β-globin gene cluster. [Copyright &y& Elsevier]

Abstract: Extracellular ATP (eATP) induces an intracellular Ca2+ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca2+ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca2+ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca2+ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca2+ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca2+ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca2+-ATPase (PMCA) or the plasma membrane Na+/Ca2+ exchanger (NCX) by LaCl3 or by replacement of Na+ with N-Methyl-Glucosamine. eATP-induced nuclear Ca2+ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca2+ transients by mobilizing a nuclear Ca2+ store via nuclear IP3Rs. [Copyright &y& Elsevier]

FK506 binding protein 12.6 kDa (FKBP12.6), a protein that regulates ryanodine Ca(2+) release channels, may act as an important regulator of insulin secretion. In this study, the role of FKBP12.6 in the control of insulin secretion and blood glucose is clarified using FKBP12.6(-/-) mice. FKBP12.6(-/-) mice showed significant fed hyperinsulinemia but exhibited normoglycemia, fasting normoinsulinemia, and normal body weight compared with wild-type (WT) littermate control mice. Deletion of FKBP12.6 resulted in enhanced glucose-stimulated insulin secretion (GSIS) both in vivo and in vitro, a result that is due to enhanced glucose-induced islet Ca(2+) elevation. After a high-fat dietary challenge (HF diet) for 3 mo, FKBP12.6(-/-) mice displayed higher body weight, hyperinsulinemia, and lower fed blood glucose concentrations compared with WT mice. FKBP12.6(-/-) mice displayed hyperinsulinemia, and resistance to HF diet-induced hyperglycemia, suggesting that FKBP12.6 plays an important role in insulin secretion and blood glucose control, and raising the possibility that it may be a potential therapeutic target for the treatment of type 2 diabetes.

The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

Extracellular ATP (eATP) induces an intracellular Ca(2+) transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca(2+) release from intracellular stores in mouse pancreatic beta-cells. Using laser scanning confocal microscopy, Ca(2+) indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca(2+) release in pancreatic beta-cell nuclei. eATP induced a higher nuclear Ca(2+) transient in pancreatic beta-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca(2+) transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca(2+)-ATPase (PMCA) or the plasma membrane Na(+)/Ca(2+) exchanger (NCX) by LaCl(3) or by replacement of Na(+) with N-Methyl-Glucosamine. eATP-induced nuclear Ca(2+) transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic beta-cells. These results indicate that eATP triggers nuclear Ca(2+) transients by mobilizing a nuclear Ca(2+) store via nuclear IP3Rs.

To delineate the relationship between epigenetic modifications and hemoglobin switching, we compared the pattern of histone acetylation and pol II binding across the beta-globin locus at fetal and adult stages of human development. To make this comparison possible, we introduced an external control into experimental samples in chromatin immunoprecipitation (ChIP) assays. Using this common standard, we found that the locus control region (LCR) was acetylated to the same level at all stages, whereas acetylation levels at the individual gene regions correlated with the state of transcription. In the active genes, the promoters were less acetylated compared with the coding regions. Furthermore, all globin promoters were acetylated to a similar level irrespective of the state of transcription. However, after correction for the loss of nucleosomes, the level of acetylation per histone at the active gamma and beta promoters was 5- to 7-fold greater than that at the inactive epsilon promoter. Although the histone acetylation level within the LCR was developmentally stable, pol II binding in fetal erythroblasts was 2- to 3-fold greater than that in adult erythroblasts. These results demonstrate that dynamic changes in histone acetylation and pol II take place as the human beta-globin gene region undergoes its developmental switches.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal gamma-globin gene results in reactivation of the gamma-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced beta locus YAC transgenic mice carrying an exact beta locus replicate of a deletional HPFH mutation, HPFH 2. Although the gamma-globin gene was expressed in the HPFH 2/beta locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of gamma-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the gamma-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the beta-YAC construct might be involved in gamma-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.

SSOs (single-stranded oligonucleotides) can mediate site-specific alteration of base-pairs in episomal and chromosomal target genes in mammalian cells. The TNE (targeted nucleotide exchange) can result in either repair or mutation of a gene sequence and is mediated through endogenous DNA repair pathway(s). Thus the approach provides a technique for the treatment of monogenic disorders associated with specific point mutations such as SCD (sickle cell disease). We studied the potential application of SSOs for SCD by introducing either an A to T substitution at the sixth codon of the human beta-globin gene (sickle locus) or a C to G mutation at -202 of the Ggamma-globin gene promoter region. The latter TNE is an alternative strategy to ameliorate the clinical manifestations of sickle cell anaemia by re-activating fetal haemoglobin gene expression in adult erythrocytes. A sensitive and valid PCR assay system was developed, which allows detection of point mutations as low as 0.01% at these sites. Using this system, TNE between 0.01 and 0.1% at the sickle locus or gamma-globin gene promoter region was detected after transfection with SSOs in cultured human cell lines. TNE in the Ggamma-globin promoter region exhibited varying degrees of strand bias that was dependent on SSO design and the cell's DNA mismatch repair activity. The results suggest that the endogenous DNA repair machinery may permit SSO correction of the sickle defect by modification of the beta- and/or gamma-globin genes.