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Background: The COVID-19 outbreak disrupted medical access for patients receiving chronic opioid therapy. This study investigated their prescription opioid dosages before and after the 2020 outbreak in Taiwan. Methods: A prospective questionnaire survey was conducted among registered outpatients receiving long-term opioids before July 2019 in Taiwan. The questionnaire included items from the Taiwanese Brief Pain Inventory and quality of life assessment. Follow-up surveys in outpatient departments through October 2020 were conducted to collect opioid prescription data. Results: After a mean of 531 days, the questionnaire responses of 103 of the initial 117 respondents were reviewed. Daily opioid doses decreased for 31 respondents (30.1%), remained roughly equivalent (defined as ±2.5%) for 27 (26.2%), and increased for 45 (43.7%) after the first wave of the pandemic. The use of strong opioids and nonopioid medications did not significantly differ among the three groups, but less fentanyl patch use was noted in the decreased-dose group after the outbreak. More than 70% of the patients received daily high-dose opioids (≥90 morphine milligram equivalents); moreover, 60% reported constipation. No deaths due to opioid overdose occurred during the study period. Conclusions: The COVID-19 outbreak in 2020 did not interrupt access to long-term opioid prescriptions for most registered patients with chronic pain in Taiwan. Less fentanyl patch use was observed in participants whose opioid dose was tapering.

Background: Erector spinae plane block could be a potential alternative to paravertebral block or other analgesic techniques for breast surgery, but the current evidence on erector spinae plane block in breast surgery is conflicting. Objective: To compare the analgesic effectiveness between erector spinae plane block, systemic analgesic, and paravertebral block for breast surgery. Study Design: Meta-analysis. Setting: The literature search was performed from 2016 to August 2020 using the MEDLINE, EMBASE, Cochrane library, and ClinicalTrials.gov databases. Methods: Clinical trials comparing erector spinae plane block to systemic analgesic and paravertebral block were included from the aforementioned databases. Primary outcomes were 24-hour postoperative opioid administration and postoperative pain score. Secondary outcomes were patient satisfaction levels, post-anesthesia care unit and hospital stay, block-related side effects, and opioid-related side effects. Systematic search, critical appraisal, and pooled analysis were performed according to the PRISMA statement. Results: We analyzed 495 cases in 8 randomized controlled trials. Compared with a systemic analgesic, the use of erector spinae plane block resulted in a reduced 24-hour postoperative intravenous morphine equivalent dose by a mean difference of 7.59 mg (P < 0.00001). Compared with paravertebral block, no statistical difference was found in opioid administration. No differences were observed in pain score, opioid-related side effects, or analgesic technique-related complications. Between the trials, heterogeneity existed and could not be evaluated using metaregression owing to inadequate reported data. Limitations: Moderate heterogeneity among the included trials could not be assessed by potential covariates owing to the limited reported data in each trial. Conclusion: Erector spinae plane block is superior to systemic analgesic within 24 hours after breast surgery and can serve as an alternative to paravertebral block with similar analgesic effects. Key words: Erector spinae plane block, paravertebral block, breast surgery, perioperative analgesia, randomized controlled trial, meta-analysis

The World Health Organization has highlighted the importance of an international standard (IS) for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) neutralizing antibody titer detection to calibrate diagnostic techniques. We applied an IS to calibrate neutralizing antibody titers (NTs) (international units/mL) in response to coronavirus disease 2019 (COVID-19) vaccination. Moreover, the association between different factors and neutralizing antibodies was analyzed. A total of 1667 serum samples were collected from participants receiving different COVID-19 vaccines. Antibody titers were determined by a microneutralization assay using live viruses in a biosafety level 3 (BSL-3) laboratory and a commercial serological MeDiPro kit. The titer determined using the MeDiPro kit was highly correlated with the NT determined using live viruses and calibrated using IS. Fever and antipyretic analgesic treatment were related to neutralizing antibody responses in ChAdOx1-S and BNT162b2 vaccinations. Individuals with diabetes showed a low NT elicited by MVC-COV1901. Individuals with hypertension receiving the BNT162b2 vaccine had lower NTs than those without hypertension. Our study provided the international unit (IU) values of NTs in vaccinated individuals for the development of vaccines and implementation of non-inferiority trials. Correlation of the influencing factors with NTs can provide an indicator for selecting COVID-19 vaccines based on personal attributes.

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in retinoic acid-inducible gene I (RIG-I) translocation to mitochondrial antiviral-signaling protein (MAVS) to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-β promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-β expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C terminus-truncated NS1 with a T49A mutation dramatically increases IFN-β mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-β expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in retinoic acid-inducible gene I (RIG-I) translocation that induces type I interferon (IFN) expression, and that NS1 protein prevents RIG-I translocation to the mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-β suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

PT-2385 is currently regarded as a potent and selective inhibitor of hypoxia-inducible factor-2α (HIF-2α), with potential antineoplastic activity. However, the membrane ion channels changed by this compound are obscure, although it is reasonable to assume that the compound might act on surface membrane before entering the cell´s interior. In this study, we intended to explore whether it and related compounds make any adjustments to the plasmalemmal ionic currents of pituitary tumor (GH3) cells and human 13-06-MG glioma cells. Cell exposure to PT-2385 suppressed the peak or late amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner, with IC50 values of 8.1 or 2.2 µM, respectively, while the KD value in PT-2385-induced shortening in the slow component of IK(DR) inactivation was estimated to be 2.9 µM. The PT-2385-mediated block of IK(DR) in GH3 cells was little-affected by the further application of diazoxide, cilostazol, or sorafenib. Increasing PT-2385 concentrations shifted the steady-state inactivation curve of IK(DR) towards a more hyperpolarized potential, with no change in the gating charge of the current, and also prolonged the time-dependent recovery of the IK(DR) block. The hysteretic strength of IK(DR) elicited by upright or inverted isosceles-triangular ramp voltage was decreased during exposure to PT-2385, meanwhile, the activation energy involved in the gating of IK(DR) elicitation was noticeably raised in its presence. Alternatively, the presence of PT-2385 in human 13-06-MG glioma cells effectively decreased the amplitude of IK(DR). Considering all of the experimental results together, the effects of PT-2385 on ionic currents demonstrated herein could be non-canonical and tend to be upstream of the inhibition of HIF-2α. This action therefore probably contributes to down-streaming mechanisms through the changes that it or other structurally resemblant compounds lead to in the perturbations of the functional activities of pituitary cells or neoplastic astrocytes, in the case that in vivo observations occur.

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the μ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na+ current (INa ) with an IC50 value of 1.9 μM. It shifted the steady-state inactivation curve of peak INa to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak INa . In continued presence of NAL, subsequent application of either dynorphin A1-13 (1 μM) or naloxone (30 μM) failed to modify its suppression of peak INa . Tefluthrin (Tef; 10 μM), a pyrethroid known to activate INa , increased peak INa with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak INa followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K+ current [IK(M) ] with an IC50 value of 5.7 μM, while it slightly suppressed erg-mediated and delayed-rectifier K+ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca2+ -activated K+ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak INa , and subsequent addition of Tef reversed NAL-induced suppression of INa . Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including INa and IK(M) , thereby influencing the functional activities of central neurons.

Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (Ih) and also reported to ameliorate inflammatory or neuropathic pain. However, to what extent this agent can perturb another types of membrane ion currents in neurons or endocrine cells remains to be largely unknown. Therefore, the Ih or other types of ionic currents in pituitary tumor (GH3) cells and in hippocampal mHippoE-14 neurons was studied with or without the presence of IVA or other related compounds. The IVA addition caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 0.64 μM and a KD value of 0.68 μM. IVA (0.3 μM) shifted the Ih activation curve to a more negative potential by approximately 8 mV, despite no concomitant change in the gating charge. Additionally, IVA was found to increase M-type K+ current (IK(M)) together with a rightward shift in the activation curve. In cell-attached current recordings, IVA (3 μM) applied to the bath increased the open probability of M-type K+ channels; however, it did not modify single-channel conductance of the channel. In current-clamp voltage recordings, IVA suppressed the firing of spontaneous action potentials in GH3 cells; and, further addition of linopirdine attenuated its suppression of firing. In hippocampal mHippoE-14 neurons, IVA also effectively increased IK(M) amplitude. In summary, both inhibition of Ih and activation of IK(M) caused by IVA can synergistically combine to influence electrical behaviors in different types of electrically excitable cells occurring in vivo.

Parecoxib, a prodrug of valdecoxib, is a selective inhibitor of cyclooxygenase-2 and widely used for traumatic and postoperative patients to avoid opioid-induced side effects. It is a potent analgesic and has a role in multimodal analgesic and enhanced recovery after surgery. Whether parecoxib exerts any actions on these types of ionic currents remains unclear. In this study, we investigated whether it exerts any effects on ion currents in differentiated NG108-15 neuronal cells. Cell exposure to parecoxib (1–30 μM) caused a reversible reduction in the amplitude of IK(DR) with an IC50 value of 9.7 μM. The time course for the IK(DR) inactivation in response to a long-lasting pulse was changed to the biexponential process during cell exposure to 3 μM parecoxib. Other agents known to inhibit the cyclooxygenase activity have minimal effects on IK(DR). Parecoxib enhanced the degree of excessive accumulative inhibition of IK(DR) inactivation evoked by a train of brief repetitive stimuli. This compound suppressed the amplitude of M-type K+ current. It depressed the peak amplitude of voltage-gated Na+ current with no change in the current-voltage relationship of this current. However, it did not have any effect on hyperpolarization-activated cation current. No change in the expression level of KV3.1 mRNA was detected in the presence of parecoxib. The effects of parecoxib on ion currents are direct and unrelated to its inhibition of the enzymatic activity of cyclooxygenase-2. The inhibition of these ion channels by parecoxib may partly contribute to the underlying mechanisms by which it affects neuronal function in vivo.

Background: Long-term use of opioids for chronic noncancer pain is associated with sex hormone disturbances. The interferences with sex hormones, sexual function, and depression were investigated in patients with chronic noncancer pain. Methods: A cross-sectional multicenter survey was conducted on 170 officially registered outpatients receiving long-term opioid treatment in nine medical centers in Taiwan between October 2018 and July 2019. Serum sex hormone levels were examined after the collection of self-administered questionnaires containing the Taiwanese version of the Brief Pain Inventory, depressive status, and sexual function interference. Results: Among 117 (68.8%) questionnaire responses from 170 enrolled outpatients, 38 women and 62 men completed the sex hormone tests, among whom only 23 (23%) had previously received blood hormone tests. Low serum total testosterone levels were detected in 34 (89.5%) women (&lt, 30 ng/dL) and 31 (50%) men (&lt, 300 ng/dL). Over 60% of women and men reported reduced sexual desire and function despite a nearly 50% reduction in pain intensity and daily function interference over the previous week after opioid treatment. Women generally had higher risks of a depression diagnosis (p = 0.034) and severe depressive symptoms (p = 0.003) and nonsignificantly lower opioid treatment duration (median 81 vs. 120 months) and morphine milligram equivalent (median 134 vs. 165 mg/day) compared with men. Conclusions: This survey demonstrated the high prevalence of depression diagnosis, low sex hormone levels, and reduced sexual function among Taiwanese patients with chronic noncancer pain receiving prolonged opioid therapy. Regular hypogonadal screenings are recommended for further management.

Sleep apnea disrupts physiologic homeostasis and causes neuronal dysfunction. In addition to signs of mental disorders and cognitive dysfunction, patients with sleep apnea have a higher anxiety rate. Here, we examined the mechanisms underlying this critical health issue. We used a mouse model with sleep-associated chronic intermittent hypoxia (IH) to verify the effects of sleep apnea on neuronal dysfunction. To evaluate how IH alters neuronal function to yield anxiety-like behavior and cognitive dysfunction, we examined synaptic plasticity and neuronal inflammation in related brain areas, including the medial prefrontal cortex (mPFC), striatum, and hippocampus. Mice subjected to chronic IH for 10 days exhibited significant anxiety-like behaviors in the elevated plus maze test. IH mice spent less travel time in open arms and more travel time in enclosed arms compared to control mice. However, cognitive impairment was minimal in IH mice. Increased glutamate N-methyl-D-aspartate (NMDA) receptor subunits 2B (GluN2B) and phosphorylated-ERK1/2 were seen in the mPFC, striatum, and hippocampus of IH mice, but no significant microglial and astrocyte activation was found in these brain areas. Chronic IH in mice induced compensatory increases in GluN2B to disturb neuronal synaptic plasticity, without neuronal inflammation. The altered synaptic plasticity subsequently led to anxiety-like behavior in mice. Treatment with the NMDA receptor antagonist dextromethorphan attenuated chronic IH-induced anxiety-like behavior and GluN2B expression. Our findings provide mechanistic evidence of how IH may provoke anxiety and support for the importance of early intervention to alleviate anxiety-associated complications in patients with chronic sleep apnea.

Epidemiologic studies have indicated that chronic hypertension may facilitate the progression of abnormal behavior, such as emotional irritability, hyperactivity, and attention impairment. However, the mechanism of how chronic hypertension affects the brain and neuronal function remains unclear. In this study, 58-week-old male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) control rats were used. Their locomotor activity and neuronal function were assessed by the open field test, novel object, and Y maze recognition test. Moreover brain tissues were analyzed. We found that the aged SHR exhibited significant locomotor hyperactivity when compared to the WKY rats. However, there was no significant difference in novel object and novel arm recognition between aged SHR and the WKY rats. In the analysis of synaptic membrane protein, the expression of glutamatergic receptors, such as the N-methyl-D-aspartate (NMDA) receptor receptors subunits 2B (GluN2B) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 1 (GluA1) in the hippocampus of SHR were significantly higher than those of WKY rats. In addition, in the synaptic membrane of SHR's hippocampus and medial prefrontal cortex (mPFC), a down-regulation of astrocytes was found, though the excitatory amino acid transporter 2 (EAAT2) remained constant. Moreover, a down-regulation of microglia in the hippocampus and mPFC was seen in the SHR brain. Long-term exposure to high blood pressure causes upregulation of glutamate receptors. The upregulation of glutamatergic receptors in hippocampus may contribute to the hyper-locomotor activity of aged rodents and may as a therapeutic target in hypertension-induced irritability and hyperactivity.

Background Huntington's disease (HD) is an inherited disease characterized by both mental and motor dysfunctions. Our previous studies showed that HD mice demonstrate a diminished pain response. However, few studies have focused on the relationship between HD and morphine analgesia. The purpose of this study is to investigate and compare the analgesic effects of morphine in HD and wild-type (WT) mice. Methods We used clinically similar transgenic HD mice (7-10 weeks of age with motor dysfunction at 8-9 mo of age) carrying a mutant Huntington CAG trinucleotide repeats to evaluate morphine analgesia. The morphine (10 mg/kg subcutaneously) analgesia was evaluated with a tail-flick in hot water (52°C). Mice spinal cords were harvested at the end of the analgesia studies. An immunofluorescence assay and western blotting were used to identify changes in the cells and cytokines. Results Our data demonstrate that preonset young HD mice exhibited a better analgesic response to morphine than the WT mice. Western blotting and an immunohistological examination of the lumbar spinal cord tissue indicated less activation of glial cells and astrocytes in the HD mice compared with the WT mice. The production levels of tumor necrosis factor α and interleukine-1β were also lower in the young HD mice. Conclusion Our data demonstrate better morphine analgesic and less pain-related cytokine responses at the spinal cord level for HD mice. Further studies are needed to determine the morphine analgesia mechanism in HD.

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin disease characterized by blisters and ulcers on the skin and mucosa after minor friction. The risk of invasive squamous cell carcinoma on the unhealed ulcers increases with age. Tracheal intubation during general anesthesia may induce tracheal stricture due to blister formation and/or scarring in DEB patients and cause severe airway obstruction. There is no consensus for handling DEB patients' fragile mucosa and skin during general anesthesia. We report an adult DEB patient who received two operations under different general anesthesia methods. The experience from this particular patient and her response to anesthesia may provide a satisfactory guide to avoid complications and improve the outcome for DEB patients receiving general anesthesia.

Sleep apnea disrupts physiologic homeostasis and causes neuronal dysfunction. In addition to signs of mental disorders and cognitive dysfunction, patients with sleep apnea have a higher anxiety rate. Here, we examined the mechanisms underlying this critical health issue. We used a mouse model with sleep-associated chronic intermittent hypoxia (IH) to verify the effects of sleep apnea on neuronal dysfunction. To evaluate how IH alters neuronal function to yield anxiety-like behavior and cognitive dysfunction, we examined synaptic plasticity and neuronal inflammation in related brain areas, including the medial prefrontal cortex (mPFC), striatum, and hippocampus. Mice subjected to chronic IH for 10 days exhibited significant anxiety-like behaviors in the elevated plus maze test. IH mice spent less travel time in open arms and more travel time in enclosed arms compared to control mice. However, cognitive impairment was minimal in IH mice. Increased glutamate N-methyl-D-aspartate (NMDA) receptor subunits 2B (GluN2B) and phosphorylated-ERK1/2 were seen in the mPFC, striatum, and hippocampus of IH mice, but no significant microglial and astrocyte activation was found in these brain areas. Chronic IH in mice induced compensatory increases in GluN2B to disturb neuronal synaptic plasticity, without neuronal inflammation. The altered synaptic plasticity subsequently led to anxiety-like behavior in mice. Treatment with the NMDA receptor antagonist dextromethorphan attenuated chronic IH-induced anxiety-like behavior and GluN2B expression. Our findings provide mechanistic evidence of how IH may provoke anxiety and support for the importance of early intervention to alleviate anxiety-associated complications in patients with chronic sleep apnea.

Several studies have demonstrated increased postoperative mortality rates in patients on chronic hemodialysis compared with non-dialyzed patients. However, limited studies have examined factors that may contribute to postoperative mortality.In this retrospective cohort study, data were collected from 9,140 dialysis and 45,725 non-dialysis patients undergoing surgery between 2007 to 2009 from Taiwan's National Health Insurance Registry Database. Patient demographics, comorbidities, and anesthesia duration were used to compare 30-day postoperative mortality differences in dialysis patients.Dialysis patients undergoing first-time surgery were significantly older, more likely male, and possessed more comorbidities. Overall, dialysis patients had significantly higher all-cause postoperative mortality (odds ratio, 15.005; 95% confidence interval, 11.917-18.893). Gender (hazard ratio [HR], 0.762), age (HR, 1.012), longer duration of inhalation general anesthesia (HR, 1.113), and comorbidities of hypertension (HR, 0.759), diabetes (HR, 1.339), congestive heart failure (HR, 1.232), coronary artery disease (HR, 1.326), cerebral vascular accident (HR, 1.312), intracranial hemorrhage (HR, 6.765), gastrointestinal bleeding (HR, 1.396), and liver cirrhosis (HR, 2.027), independently increased postoperative mortality risk in dialysis patients. Of the comorbidities, intracranial hemorrhage posed the greatest risk.Patient demographics, anesthesia factors, and comorbidities help dialysis patients understand their postoperative mortality. These potential risk factors also inform anesthesiologists and surgeons weight perioperative conditions in dialysis patients before surgery.

Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

The novel human coronavirus disease COVID-19 has become the fifth documented pandemic since the 1918 flu pandemic. COVID-19 was first reported in Wuhan, China, and subsequently spread worldwide. The coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses based on phylogenetic analysis. SARS-CoV-2 is believed to be a spillover of an animal coronavirus and later adapted the ability of human-to-human transmission. Because the virus is highly contagious, it rapidly spreads and continuously evolves in the human population. In this review article, we discuss the basic properties, potential origin, and evolution of the novel human coronavirus. These factors may be critical for studies of pathogenicity, antiviral designs, and vaccine development against the virus.

Croton is an extensive flowering plant genus in the spurge family, Euphorbiaceae. Three croton compounds with the common ent-kaurane skeleton have been purified from Croton tonkinensis. Methods: We examined any modifications of croton components (i.e., croton-01 [ent-18-acetoxy-7&alpha, hydroxykaur-16-en-15-one], croton-02 [ent-7&alpha, 14&beta, dihydroxykaur-16-en-15-one] and croton-03 [ent-1&beta, acetoxy-7&alpha, dihydroxykaur-16-en-15-one] on either hyperpolarization-activated cation current (Ih) or erg-mediated K+ current identified in pituitary tumor (GH3) cells and in rat insulin-secreting (INS-1) cells via patch-clamp methods. Results: Addition of croton-01, croton-02, or croton-03 effectively and differentially depressed Ih amplitude. Croton-03 (3 &mu, M) shifted the activation curve of Ih to a more negative potential by approximately 11 mV. The voltage-dependent hysteresis of Ih was also diminished by croton-03 administration. Croton-03-induced depression of Ih could not be attenuated by SQ-22536 (10 &mu, M), an inhibitor of adenylate cyclase, but indeed reversed by oxaliplatin (10 &mu, M). The Ih in INS-1 cells was also depressed effectively by croton-03. Conclusion: Our study highlights the evidence that these ent-kaurane diterpenoids might conceivably perturb these ionic currents through which they have high influence on the functional activities of endocrine or neuroendocrine cells.

Huntington disease (HD) affects the nervous system and leads to mental and motor dysfunction. Previous studies have shown that HD is caused by the exon 1 region of the huntingtin (HTT) gene having expanded CAG trinucleotide repeats. However, few studies have focused on the relationship between HD and pain. The purpose of this study is to investigate the relationship between HD and pain response.We used clinical similar transgenic HD mice carrying a mutant HTT exon 1 containing 84 CAG trinucleotide repeats to evaluate the relationship between HD and pain. Inflammatory pain models were induced by either formalin or complete Freund adjuvant injection over the hind paw. Spinal cord, dorsal root ganglion, and paw skin tissues were harvested at the end of the behavioral inflammatory pain studies. Immunofluorescence assay, Western blotting, and enzyme-linked immunosorbent assay were used to identify changes in cells and cytokines.Our data demonstrate that preonset HD mice exhibited less pain behavior than wild-type (WT) mice in both young (n = 11 [WT], 13 [HD]) and aged (n = 8 [WT], 9 [HD]) mice. Western blotting and immunohistological examination of lumbar spinal cord tissue and dorsal root ganglion indicate less activation of glial cells and astrocytes in young HD mice (n = 6-7) compared to that in WT mice (n = 6-7). The production levels of tumor necrosis factor-α, interleukin-1β, and substance P were also lower in young HD mice (n = 6-7).Our data demonstrate less pain behavior and pain-related cytokine response at the spinal cord level for HD mice compared to those for WT mice. Further studies are needed for determining the mechanism as to how mutant HTT leads to altered pain behavior and pain-related cytokine response.

Curcumin (CUR) has been demonstrated to induce insulin release from pancreatic β-cells; however, how curcuminoids (including demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)) exert any possible effects on membrane ion currents inherently in insulin-secreting cells remains largely unclear. The effects of CUR and other structurally similar curcuminoids on ion currents in rat insulin-secreting (INS-1) insulinoma cells were therefore investigated in this study. The effects of these compounds on ionic currents and membrane potential were studied by patch-clamp technique. CUR suppressed the amplitude of delayed-rectifier K + current ( I K(DR) ) in a time-, state- and concentration-dependent manner in these cells and the inhibition was not reversed by diazoxide, nicorandil or chlorotoxin. The value of dissociation constant for CUR-induced suppression of I K(DR) in INS-1 cells was 1.26 μM. Despite the inability of CUR to alter the activation rate of I K(DR) , it accelerated current inactivation elicited by membrane depolarization. Increasing CUR concentrations shifted the inactivation curve of I K(DR) to hyperpolarized potential and slowed the recovery of I K(DR) inactivation. CUR, DMC, and BDMC all exerted depressant actions on I K(DR) amplitude to a similar magnitude, although DMC and BDMC did not increase current inactivation clearly. CUR slightly suppressed the peak amplitude of voltage-gated Na + current. CUR, DMC and BDMC depolarized the resting potential and increased firing frequency of action potentials. The CUR-mediated decrease of I K(DR) and the increase of current inactivation also occurred in βTC-6 INS-1 cells. Taken these results together, these effects may be one of the possible mechanisms contributing their insulin-releasing effect.

The non-structural protein (NS1) of influenza A viruses (IAV) performs multiple functions during viral infection. NS1 contains two nuclear localization signals (NLS): NLS1 and NLS2. The NS1 protein is located predominantly in the nucleus during the early stages of infection and subsequently exported to the cytoplasm. A nonsense mutation that results in a large deletion in the carboxy-terminal region of the NS1 protein that contains the NLS2 domain was found in some IAV subtypes, including highly pathogenic avian influenza (HPAI) H7N9 and H5N1 viruses. We introduced different mutations into the NLS domains of NS1 proteins in various strains of IAV, and demonstrated that mutation of the NLS2 region in the NS1 protein of HPAI H5N1 viruses severely affects its nuclear localization pattern. H5N1 viruses expressing NS1 protein that is unable to localize to the nucleus are less potent in antagonizing cellular antiviral responses than viruses expressing wild-type NS1. However, no significant difference was observed with respect to viral replication and pathogenesis. In contrast, the replication and antiviral defenses of H1N1 viruses are greatly attenuated when nuclear localization of the NS1 protein is blocked. Our data reveals a novel functional plasticity for NS1 proteins among different IAV subtypes.

Few studies have investigated the effects of iron oxide nanoparticles (NPs) on analgesia. We developed inflammatory pain models via complete Freund's adjuvant injection over the hind paw in CD1 mice. Various doses of magnetite (Fe3O4) NPs were injected into the paw. Analgesia behavior was checked with von Frey microfilament and thermal irradiation measurements. Paw skin tissues were harvested at the maximal analgesia time point. The presence of activated white cells (CD68, myeloperoxidase) and free radical (reactive oxygen species, ROS) production was also checked. Western blotting was used to identify the changes of ROS production enzymes. Fe3O4 NPs demonstrated a dose-related analgesia effect with significant reduction in inflammatory cells, pro-inflammatory markers, and ROS production in the lesion paw. ROS production enzyme expression also declined. The results indicate that local Fe3O4 NP administration induced significant analgesia via attenuation of inflammatory cell infiltration and pro-inflammatory signaling as well as scavenging of microenvironment free radicals in a mouse inflammatory pain model.

Transcutaneous electrical nerve stimulators (TENSs) have been proved to be effective in muscle pain management for several decades. However, there is no consensus for the optimal TENS program. Previous research demonstrated that a 100 Hz TENS (L-TENS) provided better analgesia than a conventional TENS (< 5 Hz). However, no research compared a higher-frequency (> 100 Hz) TENS with a 100 Hz TENS. We used a 5000 Hz (5 kHz) frequency TENS (M-TENS) and an L-TENS to compare analgesic effect on a mice skin/muscle incision retraction model. Three groups of mice were used (sham, L-TENS, and M-TENS) and applied with different TENS programs on Day 4 after the mice skin/muscle incision retraction model; TENS therapy was continued as 20 min/d for 3 days. Mice analgesic effects were measured via Von Frey microfilaments with the up–down method. After therapy, mice spinal cord dorsal horn and dorsal root ganglion (DRG) were harvested for cytokine evaluation (tumor necrosis factor-α and interleukin-1β) with the Western blotting method. Our data demonstrated that the M-TENS produced better analgesia than the L-TENS. Cytokine in the spinal cord or DRG all expressed lower than that of the sham group. However, there is no difference in both cytokine levels between TENSs of different frequencies in the spinal cord and DRG. We concluded that the M-TENS produced faster and better mechanical analgesia than the L-TENS in the mice skin/muscle incision retraction model. Those behavior differences were not in accordance with cytokine changes in the spinal cord or DRG.

Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. Through genetic analysis and functional characterization, we here show that the NS segment of H7N9 virus contains a unique G540A substitution, located within a previously undefined exonic splicing enhancer (ESE) motif present in the NEP mRNA of influenza A viruses. G540A supports virus replication in mammalian cells while retaining replication ability in avian cells. Host splicing regulator, SF2, interacts with this ESE to regulate splicing of NEP/NS1 mRNA and G540A substitution affects SF2–ESE interaction. The NS1 protein directly interacts with SF2 in the nucleus and modulates splicing of NS mRNAs during virus replication. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells., Some circulating avian influenza A viruses can infect humans, but the mechanism enabling species jump is poorly understood. Here, Huang et al. identify a nucleotide in NEP of avian H7N9 viruses that affects splicing efficiency of the NS segment and supports virus replication in avian and mammalian cells.

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95&ndash, 228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the &alpha, 1 helix, &beta, 5 and &beta, 6 strands, and connecting loop between &beta, 6 and &alpha, 2 (residues 142&ndash, 178), which is the posterior part of AZ95&ndash, 228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ, amino acid residues within the anterior part of AZ95&ndash, 228 (residues 120&ndash, 145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (I

Objectives Ischemia–reperfusion (IR) features massive oxidative stress of tissues and cytokine response. Propofol and sevoflurane, both of which are commonly used anesthetics, are thought to have different antioxidant activities. The aim of this study is to delineate the influence of these two drugs on the production of free radicals and proinflammatory cytokines in IR conditions via in vitro and in vivo models. Methods An in vitro IR model was performed by incubating porcine cells (including mononuclear cells, and coronary and aortic smooth muscle cells) with either propofol 25 μM or sevoflurane 2% in the hypoxia chamber (1% O 2 , 37°C) for 1 hour, followed by room temperature air for 2 hours. Reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) were also measured via flow cytometry and enzyme-linked immunosorbent assay methods, respectively. Ten pigs were used for the in vivo study. After anesthesia with either propofol (10–15 mg/kg/h) or sevoflurane (2%), internal carotid and femoral arterial catheters were inserted for direct blood pressure monitoring and blood sampling. The IR models were produced via descending thoracic aorta clamping for 1 hour and declamping for 2 hours during the procedure for left ventricular assist device implantation. Blood serum was sampled from upper and lower body vessels for ROS and TNF-α evaluation via thiobarbituric acid reacting substances method and enzyme-linked immunosorbent assay, respectively. Results The results showed significant reduction of both ROS and TNF-α levels in the propofol group in vitro IR model. However, there was no difference in lipid peroxidation and TNF-α level between propofol and sevoflurane for the in vivo IR model. Conclusion We concluded that propofol, compared with sevoflurane, can significantly inhibit ROS formation on a cell level. In addition, propofol can significantly inhibit TNF-α formation of monocytes and coronary smooth muscle cells but not aortic smooth muscle cells.

Croton is an extensive flowering plant genus in the spurge family, Euphorbiaceae. Three croton compounds with the common ent-kaurane skeleton were purified from Croton tonkinensis. By using patch-clamp recording technique, we thoroughly examined the effect of a group of croton compounds, croton-01 (ent-18-acetoxy-7α-hydroxykaur-16-en-15-one), croton-02 (ent-7α,14β-dihydroxykaur-16-en-15-one), and croton-03 (ent-1β-acetoxy-7α,14β-dihydroxykaur-16-en-15-one), on the membrane current in SM826 and BV2 microglial cells. Although neither voltage-gated Na

Supplemental Material for Rib soft fixation produces better analgesic effects and is associated with cytokine changes within the spinal cord in a rat rib fracture model by Yuan-Yuarn Liu, Jeffrey Chi-Fei Wang, Ya-Chi Lin, Hung-Tsung Hsiao and Yen-Chin Liu in Molecular Pain

Parecoxib, a prodrug of valdecoxib, is a selective inhibitor of cyclooxygenase-2 and widely used for traumatic and postoperative patients to avoid opioid-induced side effects. It is a potent analgesic and has a role in multimodal analgesic and enhanced recovery after surgery. Whether parecoxib exerts any actions on these types of ionic currents remains unclear. In this study, we investigated whether it exerts any effects on ion currents in differentiated NG108-15 neuronal cells. Cell exposure to parecoxib (1-30 μM) caused a reversible reduction in the amplitude of I

Background Nanoparticles have become one of the most promising among the potential materials used for biomedical applications. However, few researchers have focused on their effects on analgesia. Despite the fact that various nanoparticles have been evaluated for drug delivery and MRI imaging contrast enhancement in clinical settings, no reports have investigated the in vivo synergy of ketorolac iron-oxide nanoparticle conjugates to improve the analgesic effect. Methods Ketorolac conjugated magnetic iron oxide nanoparticles (Keto-SPIO) were synthesized via two-stage additions of protective agents and chemical co-precipitation. ICR mice were used to develop inflammatory pain models induced by Complete Freund’s adjuvant (CFA) injection in the hind paw. Different magnet field strengths and polarities were applied to the spinal cord after injecting Keto-SPIO into the theca space. Analgesia behavior was evaluated with the up-down method via von Frey microfilament measurement. Spinal cord tissues were harvested at the end analgesia time point upon induction of the inflammatory pain. The presence of the two cyclooxygenases (COX) in the spinal cord was examined via Western blotting to quantify the changes after intra-thecal Keto-SPIO administration. Results Intrathecal Keto-SPIO administration demonstrated a magnetic field-dependent analgesia effect in CFA pain model with a significant reduction in COX expression. Conclusions Our results indicated that intrathecal administration of the Keto-SPIO combined magnet field modulated delivery significantly promoted an analgesia effect with suppression of COX in the mice inflammatory pain model. Electronic supplementary material The online version of this article (10.1186/s12951-018-0375-9) contains supplementary material, which is available to authorized users.

The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. Influenza viruses with NS1 deletions (DelNS1 influenza viruses) are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3′ noncoding region of the M segment of viral RNA (vRNA) which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus showed that the lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus-infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts and provide strategic information important to construction of vaccine strains with NS1 deletions. IMPORTANCE Nonstructural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and live attenuated viruses with NS1 deletions show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication-competent DelNS1 virus which carries an A14U substitution in the 3′ noncoding region of the vRNA M segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent strains with NS1 deletions for use in functional and vaccine studies.