Regulation of Dihydropyrimidinase-like 3 Gene Expression by MicroRNAs in PC12 Cells with Induced Ischaemia and Hypothermia.

Although hypothermic treatment has been reported to have some beneficial effects on ischaemia at the clinical level, the mechanism of ischaemia suppression by hypothermia remains unclear due to a lack of mechanism understanding and insufficient data. The aim of this study was to isolate and characterize microRNAs specifically expressed in ischaemia-hypothermia for the dihydropyrimidinase-like 3 (Dpysl3) gene. PC12 cells were induced with CoCl2 for chemical ischaemia and incubated at 32 ℃ for hypothermia. In ischaemia-hypothermia, four types of microRNAs (miR-106b-5p, miR-194-5p, miR-326-5p, and miR-497-5p) were highly related to the Dpysl3 gene based on exosomal microRNA analysis. Dpysl3 gene expression was up-regulated by miR-497-5p but down-regulated by miR-106b-5p, miR-194-5p and miR-326-5p. Our results suggest that these four microRNAs are involved in the regulation of Dpysl3 gene expression. These findings provide valuable clues that exosomal microRNAs could be used as therapeutic targets for effective treatment of ischaemia.

Induction of apoptosis through the up-regulation of endoplasmic reticulum stress sensors by 5-hydroxy-7- (4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone.

The diarylheptanoid, 5-hydroxy-7-(4"-hydroxy-3"-metho-xyphenyl)-1-phenyl-3-heptanone (HPH), is isolated from rhizomes of Alpinia officinarum. There is no reported biological function for this compound other than the inhibition of pancreatic lipase. Cell viability, the expression of endoplasmic reticulum (ER) stress genes, the activation of ER stress sensors, and the induction of apoptosis and autophagy were confirmed following HPH treatment of PC12 cells. No cytotoxicity was observed when the cells were treated with 50 µg/ml HPH, but 40% cell death was observed using MTT assays with 100 µg/ml HPH. Although HPH did not change the expression of the ER chaperones PDI, binding BiP, and calnexin, it upregulated the expression of genes for the ER stress sensors ATF6, eIF2α, and PERK. HPH also induced apoptosis via the activation of ATF6 fragmentation, the phosphorylation of eIF2α, and XBP1 mRNA splicing. Eventually, the results of this study demonstrated that HPH induces apoptosis through upregulation of gene expression of ER stress sensors, which may provide a basis for the development of new drugs using HPH.

Impact of Endoplasmic Reticulum Stress Sensors on Pectolinarin Induced Apoptosis.

Pectolinarin, [5,7-Dihydroxy 4',6-dimethoxyflavone 7-rutinoside, 7-[[6-O-(6-Deoxy-α-L-mannopyranosyl)-ß-D-glucopyranosyl] oxy]-5-hydroxy-6-methoxy-2-(4-ethoxyphenyl)-4H-1-benzopyran-4-one], has been stated one of the major compounds in Cirsium nipponicum (Maxim.) Makino. It is characterized by biological functions of hepatoprotective, anti-inflammatory and antiobesity activities. In this research, it was explained that pectolinarin causes apoptosis in PC12 cells conducted by DNA fragmentation and formation on apoptotic bodies through the activation of ER stress sensors (ATF6 fragmentation and eIF2α phosphorylation). The result of treating the PC12 cells with 50 µM pectolinarin for 24 h has come to increase ATF6 mRNA expression up to 1.6 times, PERK expression up to 1.7 times and IRE1 expression up to 1.4 times, respectively, compared to those of the control. ATF6 fragmentation by pectolinarin treatment was increased about 2 times compared with its control, and phosphorylation of eIF2α was increased 2.5 times. The results proposed that the perception of the molecular mechanisms underlying pectolinarin-caused apoptosis may be useful in new natural medicinal products and health supplements for the apoptosis-related diseases.

Author Correction: CD200R/Foxp3-mediated signalling regulates microglial activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Higher Acid-Base Imbalance Associated with Respiratory Failure Could Decrease the Survival of Patients with Scrub Typhus during Intensive Care Unit Stay: A Gene Set Enrichment Analysis.

Ninety percent of patients with scrub typhus (SC) with vasculitis-like syndrome recover after mild symptoms; however, 10% can suffer serious complications, such as acute respiratory failure (ARF) and admission to the intensive care unit (ICU). Predictors for the progression of SC have not yet been established, and conventional scoring systems for ICU patients are insufficient to predict severity. We aimed to identify simple and robust indicators to predict aggressive behaviors of SC. We evaluated 91 patients with SC and 81 non-SC patients who were admitted to the ICU, and 32 cases from the public functional genomics data repository for gene expression analysis. We analyzed the relationships between several predictors and clinicopathological characteristics in patients with SC. We performed gene set enrichment analysis (GSEA) to identify SC-specific gene sets. The acid-base imbalance (ABI), measured 24 h before serious complications, was higher in patients with SC than in non-SC patients. A high ABI was associated with an increased incidence of ARF, leading to mechanical ventilation and worse survival. GSEA revealed that SC correlated to gene sets reflecting inflammation/apoptotic response and airway inflammation. ABI can be used to indicate ARF in patients with SC and assist with early detection.

Molecular cloning of troponin i from the two-spotted cricket, Gryllus bimaculatus, and its expression pattern during starvation / Clonagem molecular da Troponina I do grilo africano, Gryllus bimaculatus, e seu padrão de expressão durante a inanição

There is few information about troponin gene expression by starvation in insect skeletal muscle and digestive tracts. The objective of this study was to perform molecular cloning of troponin I from the two-spotted cricket, Gryllus bimaculatus (GrybiTnI) and determine its expression patterns in three different skeletal muscles and digestive tracts during starvation. GrybiTnI was translated into a protein encoding 198 amino acids with a theoretical isoelectric point of 9.78 and a molecular weight of 23671.46 Da. The GrybiTnI has both the TnC-binding site and actin/TnC-binding site shown in the typical TnI amino acid sequences. Homology analysis revealed that GrybiTnI exhibited high similarity at the amino acid level to those of other insects already reported; 89~77% identity with those of other insects. Expression of GrybiTnI by starvation did not change in dorsal wing flight muscle and dorsal ventral flight muscle, but showed up-expression in dorsal longitudinal flight muscle. In the digestive tracts, the up-expression of GrybiTnI by starvation was observed only in the hindgut but not in the rest parts including Malpighian tubules. Re-feeding following starvation restored those expressions about the level before starvation in the dorsal longitudinal flight muscle and hindgut. In conclusion, troponin modulates gene expression not only to muscle elements but also to physiological changes such as strains.

Existe pouca informação sobre a expressão gênica da troponina por inanição no músculo esquelético de insetos e no trato digestório. O objetivo deste estudo foi realizar clonagem molecular da troponina I do grilo africano, Gryllus bimaculatus (GrybiTnI) e determinar seus padrões de expressão em três diferentes músculos esqueléticos e tratos digestivos durante a inanição. GrybiTnI foi traduzido em uma proteína codificando 198 aminoácidos com um ponto isoelétrico teórico de 9,78 e um peso molecular de 23671,46 Da. O GrybiTnI tem o local de ligação a TnC e o local de ligação a actina/TnC mostrado nas sequências de aminoácidos TnI típicas. A análise de homologia revelou que GrybiTnI exibiu alta similaridade no nível de aminoácidos em relação àqueles de outros insetos já relatados; 89~77% de identidade com os de outros insetos. A expressão de GrybiTnI pela inanição não se alterou no músculo de vôo da asa dorsal e no músculo de vôo ventral dorsal, mas mostrou expressão positiva no músculo de vôo longitudinal dorsal. Nos tratos digestivos, a expressão positiva de GrybiTnI por inanição foi observada apenas no intestino grosso, mas não nas partes de repouso, incluindo os túbulos de Malpighi. A realimentação após a inanição restaurou as expressões aproximadamente ao nível antes da inanição no músculo de vôo longitudinal dorsal e no intestino grosso. Em conclusão, a troponina modula a expressão gênica não apenas em elementos musculares, mas também em alterações fisiológicas, como as cepas.

Luteolin-induced apoptosis through activation of endoplasmic reticulum stress sensors in pheochromocytoma cells.

Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] is an active flavonoid compound from Lonicera japonica (Caprifoliaceae). Luteolin inhibits tumor cell proliferation, inflammatory and oxidative stress better, when compared with other flavonoids. In the present study, it was demonstrated that luteolin induces typical apoptosis in PC12 cells (derived from a pheochromocytoma of the rat adrenal medulla) accompanied by DNA fragmentation and formation of apoptotic bodies. In addition, luteolin regulates expression of the endoplasmic reticulum (ER) chaperone binding immunoglobulin protein, activating ER stress sensors (eukaryotic initiation factor 2α phosphorylation and X­box binding protein 1 mRNA splicing) and induced autophagy. The results indicated that luteolin induces the upregulation of the unfolded protein response pathway through the ER stress sensors, which helps as an influential regulator for the apoptosis pathway in PC12 cells. The results suggested that the understanding of the molecular mechanisms underlying luteolin­induced apoptosis may be useful in cancer therapeutics, chemoprevention and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease.

CD200R/Foxp3-mediated signalling regulates microglial activation.

The heterogeneity of microglial functions have either beneficial or detrimental roles in specific physiological or pathological environments. However, the details of what transcriptional mechanisms induce microglia to take beneficial phenotypes remain unknown. Here, we report that Foxp3 is essential for beneficial outcome of the microglial response and depends upon signalling by the immunoglobulin CD200 through its receptor (CD200R). Foxp3 expression was up-regulated in microglia activated by excitotoxicity-induced hippocampal neuroinflammation. Suppression of CD200R prevented anti-inflammatory phenotype of microglia, but over-expression of Foxp3 enhanced it. Phosphorylation of STAT6, a downstream effector of CD200R, modulated transcription of Foxp3. Finally, CD200R/Foxp3-mediated signalling enhanced hippocampal neuronal viability and conferred a degree of neuroprotection, presumably by counteracting inducible nitric oxide synthase. We conclude that enhancement of Foxp3 through CD200R could be neuroprotective by targeting the microglia.

Photoexcited Porphyrins as a Strong Suppressor of ß-Amyloid Aggregation and Synaptic Toxicity.

The abnormal assembly of ß-amyloid (Aß) peptides into neurotoxic, ß-sheet-rich amyloid aggregates is a major pathological hallmark of Alzheimer's disease (AD). Light-induced photosensitizing molecules can regulate Aß amyloidogenesis. Multiple photochemical analyses using circular dichroism, atomic force microscopy, dot blot, and native gel electrophoresis verified that photoactivated meso-tetra(4-sulfonatophenyl)porphyrin (TPPS with M = 2H(+), Zn(2+), Cu(2+), Mn(2+)) successfully inhibits Aß aggregation in vitro. Furthermore, Aß toxicity was relieved in the photoexcited-TPPS-treated Drosophila AD model. TPPS suppresses neural cell death, synaptic toxicity, and behavioral defects in the Drosophila AD model under blue light illumination. Behavioral phenotypes, including larval locomotion defect and short lifespan caused by Aß overexpression, were also rescued by blue light-excited TPPS.

Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.

Low-dose radiation suppresses Pokemon expression under hypoxic conditions.

Our previous data demonstrated that CoCl2-induced hypoxia controls endoplasmic reticulum (ER) stress-associated and other intracellular factors. One of them, the transcription factor Pokemon, was differentially regulated by low-dose radiation (LDR). There are limited data regarding how this transcription factor is involved in expression of the unfolded protein response (UPR) under hypoxic conditions. The purpose of this study was to obtain clues on how Pokemon is involved in the UPR. Pokemon was selected as a differentially expressed gene under hypoxic conditions; however, its regulation was clearly repressed by LDR. It was also demonstrated that both expression of ER chaperones and ER stress sensors were affected by hypoxic conditions, and the same results were obtained when cells in which Pokemon was up- or down-regulated were used. The current state of UPR and LDR research associated with the Pokemon pathway offers an important opportunity to understand the oncogenesis, senescence, and differentiation of cells, as well as to facilitate introduction of new therapeutic radiopharmaceuticals.

Naloxone induces endoplasmic reticulum stress in PC12 cells.

Naloxone is an opioid inverse agonist used in the treatment of opiate overdose, with well known pharmacology. In the present study, we determined the effects of naloxone on the unfolded protein response (UPR) in PC12 cells. Data from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that naloxone may accelerate PC12 cell apoptosis in a dose-dependent manner. We also demonstrated that naloxone upregulated gene expression of endoplasmic reticulum (ER) chaperones, including binding immunoglobulin protein (Bip), calnexin, ER protein 29 (ERp29) and protein disulfide isomerase (PDI), and ER stress sensors, including activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase-like ER kinase (PERK). In addition, naloxone also induced typical ER stress phenomena, including ART6 proteolytic cleavage, eIF2α phosphorylation and XBP1 mRNA splicing. Furthermore, naloxone upregulated gene expression of ER chaperones and ER stress sensors in in vivo experiments. To the best of our knowledge, these results are the first to indicate that naloxone induces ER stress in vitro and in vivo.

Prevertebral paraspinal inflammation presenting with acute abdominal pain.

CT or MRI evaluation as a further diagnostic modality may be recommended in a patient showing vague clinical manifestation in the second order when patients present with subjective discomfort and simple and initial radiological modalities do not show any specific abnormal findings.

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals.

Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors. Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, [corrected] regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and [corrected] increased food intake. Our findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila melanogaster and mammals.

Precise minute ventilation delivery using a bag-valve mask and audible feedback.

OBJECTIVES: The efficacy of a modified bag-valve mask (BVM) with a ventilation rate alarm system was compared with conventional BVM to maximize adequate minute ventilation volume delivery in a manikin model. METHODS: After a 30-minute instructional session on how to use the 2 types of BVM, volunteers were randomly assigned to ventilate a manikin in a 2-minute arrest simulation using 2 different types of BVM. The manikin cardiopulmonary resuscitation was performed with a mechanical chest compression device, to which we added a rate alarm, which makes a ticking sound to indicate each second and buzzes every sixth second, to ensure a regular ventilation rate (10 breaths per minute). Fifty-two volunteers attempted to squeeze the classic BVM at a rate of 8 to 10 times per minute during 2-minute trial (volume marked BVM [VBVM]). After a 1-hour break, artificial ventilation was performed at a rate of 9 times per minute with the guidance of the rate alarm (rate and volume adjusted BVM [RVBVM]). RESULTS: There were no correlations between the data and the participants' physical characteristics or levels of training. In this study, the accuracy of minute ventilation between the 2 groups showed a significant difference (P < .001). The minute ventilation rate was constant in the RVBVM group, whereas in the VBVM group, the minute ventilation rate was irregular. CONCLUSION: In a manikin arrest model, the use of RVBVM results in a more constant and regular minute tidal ventilation rate than the use of VBVM and is, therefore, expected to produce more favorable outcomes in practical resuscitative situations.

Mucilaginibacter angelicae sp. nov., isolated from the rhizosphere of Angelica polymorpha Maxim.

A Gram-negative-staining, non-motile rod, designated GG-w14(T), was isolated from the rhizosphere of Angelica polymorpha Maxim. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolate belonged to the genus Mucilaginibacter and exhibited 93.9-97.4% 16S rRNA gene sequence similarity with recognized members of the genus Mucilaginibacter (closest relative Mucilaginibacter gossypii Gh-67(T)). DNA-DNA relatedness between strain GG-w14(T) and M. gossypii KCTC 22380(T) was <41%. Strain GG-w14(T) grew at 4-35 °C, at pH 5.0-8.0 and with 0-1% (w/v) NaCl. The isolate hydrolysed casein, CM-cellulose and starch and contained menaquinone 7 as the major menaquinone. The major cellular fatty acids were summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH; 39.9%), iso-C(15:0) (24.2%) and iso-C(17:0) 3-OH (12.4%). The DNA G+C content was 42.5 mol%. These data suggest that strain GG-w14(T) should be considered as a representative of a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter angelicae sp. nov. is proposed. The type strain is GG-w14(T) (=KCTC 23250(T)=NCAIM B 02415(T)).

Lidocaine induces endoplasmic reticulum stress-associated apoptosis in vitro and in vivo.

We demonstrated that upregulation of both gene expression of endoplasmic reticulum (ER) stress chaperones (BiP, calnexin, calreticulin, and PDI) and ER stress sensors (ATF6, IRE1 and PERK) was induced by lidocaine, a local anesthetic, in PC12 cells. In addition to gene regulation, lidocaine also induced typical ER stress phenomena such as ART6 proteolytic cleavage, eIF2 alpha phosphorylation, and XBP1 mRNA splicing. In in vivo experiments, while lidocaine downregulated gene expression of antiapoptotic factors (Bcl-2 and Bcl-xl), pro-apoptotic factor (Bak and Bax) gene expression was upregulated. Furthermore, lidocaine induced apoptosis, as measured histochemically, and upregulated PARP1, a DNA damage repair enzyme. These results are the first to show that lidocaine induces apoptosis through ER stress in vitro and in vivo.

Silkworm hemolymph down-regulates the expression of endoplasmic reticulum chaperones under radiation-irradiation.

We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, "ischemia-responsive protein 94 kDa" (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation.