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The third opium war may have already started, not only due to illicit opioid trafficking from the Golden Crescent and Golden Triangle on the international front but also through indiscriminate opioid prescription and opioid diversion at home. Opioid use disorder (OUD), among unintentional injuries, has become one of the top 4 causes of death in the United States (U.S.). An OUD is defined as a problematic pattern of opioid use resulting in clinically significant impairment or distress, consisting of 2 or more of 11 problems within 1 year, as described by the Diagnostic and Statistical Manual for Mental Disorders, Fifth Edition. Observation of aberrant behaviors of OUD is also helpful for overworked clinicians. For the prevention of OUD, the Opioid Risk Tool and the Current Opioid Misuse Measure are appropriate screening tests before and during opioid administration, respectively. Treatment of OUD consists of 3 opioid-based U.S. Food and Drug Administration-approved medications, including methadone, buprenorphine, and naltrexone, and non-opioid-based symptomatic medications for reducing opioid withdrawal syndromes, such as α2 agonists, β-blockers, antidiarrheals, antiemetics, non-steroidal anti-inflammatory drugs, and benzodiazepines. There are at least 6 recommendable guidelines and essential terms related to OUD. Opioid stewardship programs are now critical to promoting appropriate use of opioid medications, improving patient outcomes, and reducing misuse of opioids, influenced by the successful implementation of antimicrobial stewardship programs. Despite the lack of previous motivation, now is the critical time for trying to reduce the risk of OUD.

Endocrine-disrupting chemicals (EDCs) are a structurally diverse class of synthetic and natural compounds. EDCs can cause non-communicable diseases such as obesity, type 2 diabetes, thyroid disorders, neurodevelopmental disease, hormone-dependent cancers, and reproductive disorders. The embryoid body test (EBT) is a developmental toxicity test method that determines the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The present study used the EBT to perform cytotoxicity evaluations of 10 EDCs and assessed the mechanistic relationship between endoplasmic reticulum (ER) stress and cytotoxicity. According to the statistical analysis and prediction model results, methylparaben, butylparaben, propylparaben, ethylparaben, triclosan, octylphenol, methoxychlor, bisphenol A, and diethylstilbestrol were classified as cytotoxic, but trichloroacetic acid was non-toxic. Classification accuracy was 90%. The mechanistic study showed that the cytotoxicities of butylparaben, propylparaben, octylphenol, and triclosan were induced by ER stress. The mRNA expressions of BiP, CHOP, and ATF4 were significantly higher following treatments with four EDCs compared to those after the control treatment. Compared to the control treatment, the mRNA levels of XBP1u and XBP1s increased significantly after butylparaben and propylparaben treatments, but did not increase with octylphenol and triclosan treatments. These results indicate that the EBT can be applied as an alternative toxicity test when evaluating the cytotoxicity of EDCs.

BACKGROUND The bispectral index (BIS) is the most widely used algorithm for measuring anesthetic depth. The BIS has been demonstrated as inaccurate when neuromuscular blocking drugs (NMBDs) are used. Compared with BIS, phase lag entropy (PLE), which measures the anesthetic depth based on a 4-channel EEG signal, is less affected by EMG. The purpose of this study was to compare the effect of EMG activity during emergence on anesthetic depth monitoring between PLE and BIS. METHODS Twenty five consecutive patients with physical status I-II of American Society of Anesthesiologists undergoing general anesthesia (age range, 20-60 years). The anesthesiologist attached the sensors of BIS and PLEM 100 on the patient's forehead. NMB reversal was performed by intravenously injecting sugammadex after confirmation of shallow NMB (TOF count 1-4) under neuromuscular monitoring. The BIS and PLE scores were recorded with neuromuscular monitoring at 1-min intervals for 5 min after administration of sugammadex. RESULTS The BIS and BIS-EMG measured at 1 min after sugammadex injection were significantly higher at 1 min [51.650 (46.100, 62.225) (P

The localization and expression of amylin protein in the rodent brain and mouse neuroblastoma Neuro-2a (N2a) are less widely known. Thus, this study investigated the expression distribution of amylin in the rat brain and N2a treated with steroid hormones. Amylin protein was identified in the olfactory bulb, cerebral cortex, dentate gyrus, thalamus, hypothalamus, ventral tegmental area (VTA), cerebellum, and brain stem in the rat brain. Additionally, the amylin protein was localized with the mature neurons of the cerebral cortex and dopaminergic neurons of the VTA. Progesterone (P4) and dexamethasone (Dex) significantly decreased, and 17β-estradiol (E2) increased the amylin protein level in the cerebral cortex. The P4 receptor antagonist RU486 significantly influenced the effects of P4 and Dex, and the E2 receptor antagonist ICI 182,780 slightly changed E2′s effect. Amylin protein expression was significantly reduced in the VTA by P4 and Dex, and its expression was changed only following P4 plus RU486 treatment. It was confirmed for the first time that amylin protein is strongly expressed in the cytoplasm in N2a cells using immunofluorescent staining. P4 increased the levels of amylin, and RU486 treatment decreased them. Dex significantly increased the levels of amylin protein. RU486 treatment reversed the effects of Dex. Therefore, amylin protein is expressed in the cerebral cortex neurons and dopaminergic neurons of the VTA of the immature rat brain. P4 and Dex influence the expression of amylin protein in the rat brain and N2a cells.

As people age, their risks of developing degenerative diseases such as cancer, diabetes, Parkinson’s Disease (PD), Alzheimer’s Disease (AD), rheumatoid arthritis, and osteoporosis are generally increasing. Millions of people worldwide suffer from these diseases as they age. In most countries, neurodegenerative diseases are generally recognized as the number one cause afflicting the elderly. Endoplasmic reticulum (ER) stress has been suggested to be associated with some human neurological diseases, such as PD and AD. Melatonin, a neuroendocrine hormone mainly synthesized in the pineal gland, is involved in pleiotropically biological functions, including the control of the circadian rhythm, immune enhancement, and antioxidant, anti-aging, and anti-tumor effects. Although there are many papers on the prevention or suppression of diseases by melatonin, there are very few papers about the effects of melatonin on ER stress in neurons and neurodegenerative diseases. This paper aims to summarize and present the effects of melatonin reported so far, focusing on its effects on neurons and neurodegenerative diseases related to ER stress. Studies have shown that the primary target molecule of ER stress for melatonin is CHOP, and PERK and GRP78/BiP are the secondary target molecules. Therefore, melatonin is crucial in protecting neurons and treating neurodegeneration against ER stress.

Human amylin or islet amyloid polypeptide (hIAPP) is synthesized in the pancreatic β-cells and has been shown to contribute to the pathogenesis of type 2 diabetes (T2D) in vitro and in vivo. This study compared amylin oligomerization/expression and signal transduction under endoplasmic reticulum (ER) stress and oxidative stress. pCMV-hIAPP-overexpressing INS-1E cells presented different patterns of amylin oligomerization/expression under ER stress and oxidative stress. Amylin oligomerization/expression under ER stress showed three amylin oligomers of less than 15 kDa size in pCMV-hIAPP-overexpressing cells, while one band was detected under oxidative stress. Under ER stress conditions, HIF1α, p-ERK, CHOP, Cu/Zn-SOD, and Bax were significantly increased in pCMV-hIAPP-overexpressing cells compared to the pCMV-Entry-expressing cells (control), whereas p-Akt, p-mTOR, Mn-SOD, catalase, and Bcl-2 were significantly decreased. Under oxidative stress conditions, HIF1α, p-ERK, CHOP, Mn-SOD, catalase, and Bcl-2 were significantly reduced in pCMV-hIAPP-overexpressing cells compared to the control, whereas p-mTOR, Cu/Zn-SOD, and Bax were significantly increased. In mitochondrial oxidative phosphorylation (OXPHOS), the mitochondrial complex I and complex IV were significantly decreased under ER stress conditions and significantly increased under oxidative stress conditions in pCMV-hIAPP-overexpressing cells compared to the control. The present study results demonstrate that amylin undergoes oligomerization under ER stress in pCMV-hIAPP-overexpressing cells. In addition, human amylin overexpression under ER stress in the pancreatic β cells may enhance amylin protein aggregation, resulting in β-cell dysfunction.

Rapamycin is an immunosuppressant that inhibits the mammalian or mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in organisms including mice. Myf5 and MyoD act as muscle-specific transcriptional factors for skeletal muscle differentiation. In this study, we determined whether rapamycin-induced autophagy causes the decrease of Myf5 and MyoD protein in C2C12 myoblast cells. Rapamycin induced a significant increase in the expression of the microtubule-associated protein 1 light chain 3 (LC3) II protein in a dose-dependent manner for 12 h. Rapamycin treatment also significantly increased p-ERK, p-Akt, and catalase expressions, and decreased Mn-SOD expression in a dose-dependent manner. Bax expression was significantly high compared to Bcl-2 expression in a dose-dependent manner of rapamycin for 12 h. For further study of rapamycin-induced autophagy in C2C12 myoblast cells, we investigated rapamycin treatment for 24, 36, and 48 h. Cell viability did not change with rapamycin treatment for 24, 36, and 48 h. Rapamycin-induced LC3-II, Beclin-1, Bax, and Bcl-2 proteins were significantly increased compared to without rapamycin. p-ERK expression increased with rapamycin treatment for 24 and 36 h compared to that without rapamycin, but decreased for 48 h. p-Akt expression decreased with rapamycin treatment for 36 and 48 h compared to that without rapamycin. In the same conditions, rapamycin-induced autophagy significantly reduced the Myf5 and MyoD proteins. Together, these results suggest that rapamycin-induced autophagy results in the decrease of Myf5 and MyoD proteins in C2C12 myoblast cells.

Background It is uncommon for patients who have received a permanent implant to remove the spinal cord stimulator (SCS) after discontinuation of medication in complex regional pain syndrome (CRPS) due to their completely painless state. This study evaluated CRPS patients who successfully removed their SCSs. Methods This 10-year retrospective study was performed on patients who had received the permanent implantation of an SCS and had removed it 6 months after discontinuation of stimulation, while halting all medications for neuropathic pain. Age, sex, duration of implantation, site and type of CRPS, and their return to work were compared between the removal and non-removal groups. Results Five (12.5%, M/F = 4/1) of 40 patients (M/F = 33/7) successfully removed the permanent implant. The mean age was younger in the removal group (27.2 ± 6.4 vs. 43.5 ± 10.7 years, P < 0.01). The mean duration of implantation in the removal group was 34.4 ± 18.2 months. Two of 15 patients (13.3%) and 3 of 25 patients (12%) who had upper and lower extremity pain, respectively, had removed the implant. The implants could be removed in 5 of 27 patients (18.5%) with CRPS type 1 (P < 0.01). All 5 patients (100%) who removed their SCS returned to work, while only 5 of 35 (14.3%) in the non-removal group did (P < 0.01). Conclusions Even though this study had limited data, younger patients with CRPS type 1 could remove their SCSs within a 5-year period and return to work with complete pain relief.

The embryoid body test (EBT) is a developmental toxicity test method that assesses the half inhibitory concentrations of substances in the area of embryoid bodies (EBs), and in the viability of mouse embryonic stem cells (ESCs) and fibroblasts (3T3 cells) following chemical exposure for three and four days, respectively. In the previous study, the EBT showed more advanced than the embryonic stem cell test (EST) from the European Centre for the Validation of Alternative Methods (ECVAM) applying cardiac differentiation of mouse ESCs, because the EBT greatly reduced the exposure time, labor, and amount of materials required, and misclassification of embryotoxic potential. This pre-validation study evaluated the predictive accuracy of the EBT using 26 coded test substances by two steps: intra-laboratory and inter-laboratory reproducibility tests. Since some substances have different embryotoxic potentials at different pregnancy periods, in this study, a new prediction model consisting of non-toxic and toxic classes was used, instead of the existing prediction model assessing embryotoxicants in four classes. The results of the intra- and inter-laboratory tests were highly accurate (above 80%) when substances were classified using the predictive model. In conclusion, EBT can accurately classify various embryotoxicants in a short time with less effort and greater validation.

Eun-Ji Choi,1,2 Soon Ji Park,1 Yeong-Min Yoo,1 Ji-Uk Yoon,1,2 Sang-Wook Shin,1,2 Gyeong-Jo Byeon1,2 1Department of Anesthesia and Pain Medicine, Pusan National University Yangsan Hospital, Pusan National University School of Medicine, Yangsan, Republic of Korea; 2Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Republic of KoreaCorrespondence: Gyeong-Jo ByeonDepartment of Anesthesia and Pain Medicine, Pusan National University Yangsan Hospital, Geumoro 20, Yangsan, Gyeongnam, 50612, Republic of KoreaTel +82-55-360-2129Fax +82-55-360-2149Email byeongj@pusan.ac.krBackground: Transforaminal epidural steroid injections (TF-ESIs) effectively deliver small amounts of drugs to inflamed sites via the ventral epidural space. However, there is a high risk of nerve damage as the needle narrowly approaches the spinal nerve. Therefore, we devised an oblique interlaminar (OIL) approach as an alternative method. We compared the efficacy of fluoroscopic-guided OIL-ESIs with that of TF-ESIs in the management of lower back and unilateral lumbosacral radicular pain.Materials and Methods: Sixty-six patients were randomized to receive a fluoroscopic-guided ESI either through the OIL (n = 33, group OIL) or TF (n = 33, group TF) approach. They were evaluated for effective pain relief using the visual analogue scale (VAS) and for functional improvement using the Oswestry Disability Index (ODI) and Roland Morris Disability Questionnaire (RMDQ). Other outcome measures were the presence of ventral and contralateral spread of contrast, patients’ satisfaction, and adverse events.Results: There were no significant differences between the groups in the VAS, ODI, and RMDQ scores during the 12-week period. The differences in the ODI and RMDQ scores before and after the treatment were higher in group TF than in group OIL. The contralateral spread of contrast was higher in group OIL than in group TF. There were no significant differences in the other outcomes between the groups.Conclusion: ESIs delivered through the OIL approach are equally effective in pain relief and functional improvement as those delivered via the TF approach in the management of low back and unilateral lumbosacral radicular pain.Keywords: epidural analgesia, fluoroscopy, low back pain, radicular pain, steroid injection

BACKGROUND Liver transplantation (LT) has the incidence of 30-day mortality about 5-10%, Jo et al. reported that 30-day mortality and 1-year mortality for DDLT were 30%, and 39% respectively. It is not easy to create a model for predicting post-transplantation outcomes based on pretransplant variables. MELD does not take into account individual complications such as hepatic encephalopathy (HE), and research has shown that the greater the severity of pretransplant HE, the lower the survival rate after LT; the importance of monitoring for HE is therefore emphasized. METHODS The medical records of adult patients who underwent deceased donor LT (DDLT) were retrospectively reviewed for analysis of the effect of HE on the long-term survival rate of post-transplant for more than 1 year. RESULTS Presence of HE is not statistically associated to patient survival (P=0.062), but the hazard ratio is 1.954 (95% CI, 0.968, 3.943). In addition, the severe HE group significantly decreased survival compared to the non-HE group, and the cumulative 1- and 3-year overall survival rates were 80.9% and 78.7%, respectively, in non HE group, and 65.7% and 56.1%, respectively, in severe HE group (P=0.031). CONCLUSIONS Severe HE is a factor influencing the long-term survival over 3 years in the patients who underwent DDLT. Although prospective validation should be conducted to determine the prognostic value of HE severity, efforts could be made to reduce the severity of HE before DDLT, and consider severity of HE rather than MELD score in DDLT allocation.

Background Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. Objectives This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. Methods Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. Results Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. Conclusions This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.

Background: Pancreatic beta-cell dysfunction results in reductions of insulin synthesis/secretion, cell survival, and insulin sensitivity thereby inducing diabetes mellitus. In this study, how nanomolar melatonin regulates insulin synthesis and secretion in rat insulinoma INS-1E cells was investigated. Methods: Western Blot and ELISA were performed to investigate the insulin synthesis and secretion at melatonin concentrations of 10-100 nM for 48 h and 10-500 nM for 72 h in rat insulinoma INS-1E cells.Results: Melatonin significantly increased the insulin protein level in INS-1E cells above the level in control cells without melatonin or glucose treatments and decreased the insulin protein level in media with glucose. At melatonin concentrations of 10-100 nM for 48 h and 10-500 nM for 72 h, increases and decreases occurred in dose-dependent manners. Luzindole or 4-phenyl-2-propionamidotetralin (4P-PDOT), melatonin receptor (MR) antagonists, inhibited the melatonin-induced insulin protein level in cells and media. Levels of membrane vesicle trafficking-related proteins including Rab5, GOPC, and phospho-caveolin-1 proteins significantly increased with melatonin treatment above that in control cells without melatonin or glucose treatments, whereas expressions of EEA1, clathrin, APPL1, and syntaxin-6 proteins significantly decreased with melatonin treatment. The increases in the phosphorylation of the mammalian target of rapamycin (p-mTOR) and Raptor protein levels (mTOR Complex 1 (mTORC1)) were consistent with the increments in the expressions of p-Akt (Ser473, Thr308). Also, expression levels of Bcl-2 and Bcl-xL proteins were significantly increased compared to those in control cells without melatonin or glucose treatments, whereas the Bax protein level decreased. Luzindole or 4P-PDOT decreased the melatonin-induced levels of p-mTOR (Ser2448), Raptor, p-Akt (Ser473), and Bcl-2 proteins, whereas MR antagonists increased the melatonin-suppressed level of Bax proteins. Conclusion: These results indicate that nanomolar melatonin regulates insulin synthesis and secretion associated with membrane vesicle trafficking-related proteins, including Rab5, GOPC, p-Caveolin-1, EEA1, and clathrin through the Akt/mTORC1 pathway.

The endocrine-disrupting chemical 4-tert-octylphenol (OP) is a widespread estrogenic chemical used in consumer products such as epoxy resins and polycarbonate plastic. However, the effects of OP on brain development are unknown. The present study examined the effects of OP on neuron and neurobehavioral development in mice. By using primary cortical neuron cultures, we found that OP-treated showed a decreased length of axons and dendrites and an increased number of primary and secondary dendrites. OP reduced bromodeoxyuridine (BrdU), mitotic marker Ki67, and phospho-histone H3 (p-Histone-H3), resulting in a reduction of neuronal progenitor proliferation in offspring mouse brain. Moreover, OP induced apoptosis in neuronal progenitor cells in offspring mouse brain. Furthermore, offspring mice from OP-treated dams showed abnormal cognitive, social, and anxiety-like behaviors. Taken together, these results suggest that perinatal exposure to OP disrupts brain development and behavior in mice.

Triclosan (TCS) is one of the most common endocrine-disrupting chemicals (EDCs) present in household and personal wash products. Recently, concerns have been raised about the association between abnormal behavior in children and exposure to EDC during gestation. We hypothesized that exposure to TCS during gestation could affect brain development. Cortical neurons of mice were exposed in vitro to TCS. In addition, we examined in vivo whether maternal TCS administration can affect neurobehavioral development in the offspring generation. We determined that TCS can impair dendrite and axon growth by reducing average length and numbers of axons and dendrites. Additionally, TCS inhibited the proliferation of and promoted apoptosis in neuronal progenitor cells. Detailed behavioral analyses showed impaired acquisition of spatial learning and reference memory in offspring derived from dams exposed to TCS. The TCS-treated groups also showed cognition dysfunction and impairments in sociability and social novelty preference. Furthermore, TCS-treated groups exhibited increased anxiety-like behavior, but there was no significant change in depression-like behaviors. In addition, TCS-treated groups exhibited deficits in nesting behavior. Taken together, our results indicate that perinatal exposure to TCS induces neurodevelopment disorder, resulting in abnormal social behaviors, cognitive impairment, and deficits in spatial learning and memory in offspring.

Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-DWe used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice.We found that the brains of CaBP-9k KO mice have increased APP/β-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice.Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

Essential oils are volatile compounds extracted from various plants by distillation, hydrodiffusion or compression. In recent years, the use of essential oils has gained popularity. Many pharmaceutical, cosmetic, sanitary, food industry and agriculture studies have revealed that essential oils exert antibacterial, antiviral, antifungal, antiparasitic, insecticidal, anticancer, neuroprotective, psychophysiological and anti-aging effects. Despite their reported uses, recent studies of eukaryotic cells have demonstrated that essential oils exert prooxidant and cytotoxic effects. Therefore, for the effective clinical use of essential oils, an evaluation of their cytotoxicity and the identification of the mechanisms affecting cell viability are required. To evaluate cytotoxicity, the present study determined the IC50 values of 15 essential oils provided by the Korea Forest Research Institute (Pinus densiflora for. multicaulis Uyeki, Trifolium repens, Ligularia fischeri, Abies nephrolepis, Illicium anisatum, Zanthoxylum coreanum, Abies koreana, Lindera obtusiloba, Chamaecyparis obtuse, Pinus densiflora, Magnolia kobus, Picea koraiensis, Picea abies, Abies holophylla and Platycladus orientalis). Their effect was then assessed in human lung cells (A549) and human skin cells (Detroit 551) by performing cell counting kit-8 assays. To identify the mechanism associated with each oil's cytotoxicity, expressions of cytotoxicity-associated marker genes (cyclin A, cyclin B, cyclin D and cyclin E) involved in the cell cycle and caspase-3 (involved in cell death) were examined by performing reverse transcription-quantitative PCR and western blotting. In conclusion, plant essential oils can be used as a good source of medicine. However, without examining the safety of essential oils, they cannot be used in clinics. The results included estimates of the degree of cytotoxicity and the mechanism of cell death for each oil. It is expected that the data obtained from the current study will form guidelines for the clinically appropriate and safe use of these tested essential oils.

In pancreatic β cells, which produce and secrete insulin, Ca2+ signals contribute to insulin production and secretion. Bisphenol A (BPA) and octylphenol (OP) are reported to increase plasma insulin levels and insulin transcription factors, but regulation of plasma glucose levels did not decrease proportionally to the insulin increase. We hypothesized that BPA and OP disrupt calcium homeostasis resulting in insulin resistance through induction of endoplasmic reticulum (ER) stress. BPA and OP treatment leads to survival of pancreatic β cells against streptozotocin, but despite an increased insulin level, serum glucose regulation is not properly regulated. The expression of genes involved in transporting calcium ions to the cytosol and ER decreased while the expression of those affecting the removal of calcium from the cytosol and ER increased. Depletion of calcium from the ER leads to ER stress and can induce insulin resistance. Insulin resistance is also confirmed by insulin-responsive gene, such as glucose transporter 4 (GLUT4) and IRS2, expression. Taken together, these results imply that disruption of calcium homeostasis by BPA and OP induces ER stress and leads to insulin resistance, especially in a streptozotocin (STZ) -induced type 1 diabetes mellitus model.

Streptozotocin (STZ), a glucose analog, induces diabetes in experimental animals by inducing preferential cytotoxicity in pancreatic beta cells. We investigated whether STZ reduced the production of intracellular insulin through autophagy in insulinoma INS-1E cells. Typically, 2 mM STZ treatment for 24 h significantly decreased cell survival. STZ treatment led to significant decrease in phospho-AMP-activated protein kinase (p-AMPK) level; reduction in levels of phospho-protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α); significant reduction in levels of p85α, p110, phospho-serine and threonine kinase/protein kinase B (p-Akt/PKB) (Ser473), phospho-extracellular-regulated kinase (p-ERK), and phospho-mammalian target of rapamycin (p-mTOR); increase in levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase; decrease in B-cell lymphoma 2 (Bcl-2) expression; increase in Bcl-2-associated X protein (Bax) expression; increase in levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1; and reduction in production of intracellular insulin. These results suggest that insulin synthesis during STZ treatment involves autophagy in INS-1E cells and, subsequently, results in a decrease in intracellular production of insulin.

Nitric oxide (NO) is a cellular signaling molecule in many physiological and pathological processes including neuroprotector. Here we examined the antiapoptotic effect of NO in SK-N-MC cells. H2O2 treatment (10-200 μM) induced cell death in a dose-dependent manner and pretreatment of cells with 100 μM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, attenuated the occurrence of H2O2-induced cell death. DAPI staining showed H2O2-induced nuclear fragmentation and NO treatment suppressed it. NO inhibited the proteolytic activation of caspase-3 and mitochondrial cytochrome c release. Treatment of soluble guanylyl cyclase inhibitor ODQ decreased the protective effect of SNAP on H2O2-treated cells and increased caspase 3-like enzyme activity and activation, cytochrome c release, PARP cleavage, and DNA fragmentation, indicating that cGMP is a key mediator in NO-mediated antiapoptosis. The cGMP analog 8-Br-cGMP blocked H2O2-induced apoptotic cell death; reduction of caspase-3 enzyme, cytochrome c release, and caspase-8 and -9. These preventive effects of SNAP and 8-Br-cGMP were suppressed by PKG inhibitor KT5823. Levels of PKGI, PKGII, and p-VASP proteins were increased by SNAP and 8-Br-cGMP and suppressed by KT5823 treatment. These results indicate that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Akt activation was inhibited the PI3K inhibitors LY294002 and Wortmannin, resulting in the inhibition of cell viability and increase of cytochrome c release. SNAP induced phosphorylation of Akt and Bad and then increased the interactions between 14-3-3β and p-Bad. These data suggest that the NO suppresses H2O2-induced SK-N-MC cell apoptosis by suppressing apoptosis signal mediating the interaction between 14‐3‐3β and Bad phosphorylation via PKG/PI3K/Akt.

The cytosolic calcium concentration is regulated by calcium-processing proteins such as transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). Those calcium-processing proteins are important for physiological functions in the brain. The effects of steroid hormones on calcium-processing protein expressions in the brains are unknown. Thus, the effects of steroid hormones on the distribution, localization, and expressions of calcium-processing proteins in the brain were analyzed. Immature female rats were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486). We found that TRPV5 and TRPV6 proteins were highly expressed in the cerebral cortex (CT), hypothalamus (HY), and brain stem (BS) compared to that in the olfactory bulb (OB) and cerebellum (CB). Also, the NCX1 protein was highly expressed in CT and BS compared to that in OB, HY, and CB, and PMCA1 protein was highly expressed in CT compared to that in other brain regions. Furthermore, expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and/or DEX in the CT and HY. In summary, calcium-processing proteins are widely expressed in the immature rat brain, and expressions of calcium-processing proteins in CT and HY indicated that they may regulate by E2, P4, and/or DEX and can be attenuated by antagonist treatment. These results indicate that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain.

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

Background/aims Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-D9k (CaBP-9k) acts as calcium modulators and sensors in various tissues. However, the neurobiological functions of CaBP-9k are unknown. Methods We used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice. Results We found that the brains of CaBP-9k KO mice have increased APP/β-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice. Conclusion Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.

Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.

Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.

Calbindin-D

Phytosphingosine-1-phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here, we report the expression of S1PR1-3 among P1P receptors (S1PR1-4) in cumulus cells and oocytes. When P1P was administered at concentrations of 10, 50, 100, and 1,000 nM during IVM, the metaphase II rate was significantly increased in the 1,000 nM (1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was observed only in the presence of epidermal growth factor (EGF). Oocytes under the influence of P1P showed decreased intracellular reactive oxygen species levels but no significant differences in glutathione levels. In our molecular studies, P1P treatment upregulated gene expression involved in cumulus expansion (Has2 and EGF), antioxidant enzymes (SOD3 and Cat), and developmental competence (Oct4) while activating extracellular signal-regulated kinase1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl-2 to Bax while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P displayed significantly higher developmental competence (cleavage and blastocyst [BL] formation rate) and greater BL quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization. Despite the low levels of endogenous P1P found in animals, exogenous P1P influenced animal reproduction, as shown by increased porcine oocyte maturation as well as preimplantation embryo development. This study and its findings are potentially relevant for both human and animal-assisted reproduction.

Autophagy appears to be involved in maintaining normal intracellular insulin content by accelerating the insulin degradation rate in β-cells (Marsh et al., 2007) [1]. 2-deoxy-d-glucose (2-DG) is metabolized by hexokinase, and acts as an inhibitor of glycolysis. 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways (Aghaee et al., 2012) [2], and appears to be an ideal tool for studying autophagy. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β-cells (Tanemura et al., 2012) [3]. Here, we examined that 2-DG or rapamycin-induced autophagy may decrease the production of intracellular insulin in INS-1E insulinoma cells. Data showed that autophagy was increased by 2-DG or rapamycin by Western blotting and Immunofluorescence staining analyses. Also, intracellular insulin decreased by 2-DG or rapamycin. Furthermore, the autophagy inhibitors, bafilomycin A1 and/or 3-methyladenine, in the presence or absence of 2-DG or rapamycin increased intracellular insulin in INS-1E insulinoma cells. Keywords: Insulin, Autophagy, 2-Deoxy-D-glucose, Rapamycin, INS-1E Insulinoma cells