Dimethyl Sulfoxide Control Research Articles

Cholecalciferol Exhibits no Antibacterial Effect on Staphylococcus aureus and Escherichia coli: an in-vitro Study.

The pleiotropic effect of cholecalciferol (vitamin D3) has gained significant momentum and has been explored widely. The study aimed to investigate the antimicrobial effect of cholecalciferol against S. aureus and E. coli. An in-vitro study was performed for the antimicrobial effect of cholecalciferol against S. aureus and E. coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined following the broth microdilution method. The MIC value of cholecalciferol against both S. aureus and E. coli was 0.312 mg/ml, and the MBC for both organisms was 1.25 mg/ml. However, we also observed a significant antimicrobial effect in the dimethyl sulfoxide (DMSO) control at 12.5% (v/v). Therefore, the observed antimicrobial effect may be attributed to DMSO, indicating cholecalciferol does not directly inhibit S. aureus and E. coli. This study indicates that cholecalciferol does not directly inhibit S. aureus and E. coli. Hence, we suggest exploring the antibacterial properties of other vitamin D analogs, such as calcitriol or its synergetic effect with other antimicrobial agents.

Effects of Ionizing Radiation on Intestinal Bile Acid Metabolism: Mechanism of the Radioprotective Effect of Glycoursodeoxycholic Acid

Radioactive intestinal injury is a common complication during radiotherapy of tumors. The aim of this study is to observe the effect of ionizing radiation on short-term changes in intestinal bile acids and to investigate the radioprotective effect of bile acids on intestinal cells. A rat model of small intestinal injury was constructed by exposing the abdomen of the rats to daily irradiation at 2 Gy for 4 d in succession. The bile acids were quantified using metabolomics analysis. IEC-6 cells, a small intestinal epithelial cell line, were divided into a dimethyl sulfoxide (DMSO) control group receiving DMSO and 0 Gy irradiation, a glycoursodeoxycholic acid (GUDCA) experimental group receiving GUDCA and 0 Gy irradiation, a DMSO irradiation group receiving DMSO and 10 Gy irradiation, and a GUDCA irradiation group receiving GUDCA and 10 Gy irradiation. Cell viability and cytotoxicity was assessed by CCK-8 assay test. The apoptosis rate of cells was determined by flow cytometry. The colony formation rate and the radiosensitivity of the cells were determined by colony formation assay on solid media. The expression levels of proteins associated with cell death were determined using Western blot. After exposure to irradiation, the small intestine tissues of the rats showed typical radioactive intestinal injury. In addition, various bile acids showed fluctuation before and after irradiation. Among the bile acids, GUDCA increased significantly at 3 d after irradiation, but returned to the pre-irradiation level at 7 d after irradiation. Compared with the control group, after GUDCA treatment at 20 μmol/L for 24 h, the cell viability rate after irradiation was significantly higher than that of the DMSO group (P<0.05); the expression levels of the proteins, including PARP, caspase-3, RIP, and GSDMD, were significantly lower than those in the control group (P<0.05). After GUDCA treatment at 20 μmol/L for 24 h and 48 h, the cell apoptosis rate of the cells after irradiation was lower than that of the DMSO group (P<0.05). Compared with the DMSO control group, the colony formation ability of the GUDCA experimental group was stronger than that of the DMSO group after irradiation at 0, 2, 4, and 6 Gy (P<0.05). D0, or the mean lethal dose, of the GUDCA group was 6.374, while that of the DMSO group was 4.572. Compared with the DMSO control group, the D0 value of the GUDCA treatment group increased, and the sensitization enhancement ratio (SER) was 0.717. After exposing the abdomen of rats to irradiation, the intestinal bile acid metabolism of the rats will change significantly, and GUDCA can produce radioprotective effects on intestinal cells to a certain extent.

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to PFOS, PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response () modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

Enhancing intracellular delivery of protein kinase C beta II peptide inhibitor using myristic acid-trans-activator of transcription via N-terminus conjugation in isolated rat polymorphonuclear leukocytes

Introduction: Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previous studies have shown that cell permeable myristic acid-conjugated PKCβII inhibitor (myr-PKCβII-; SLNPEWNET) (20 μM) remarkably attenuated phorbol 12-myristate 13-acetate (PMA)-induced PMN SO release when compared to scrambled control peptide (myr-PKCβII- scram; WNPESLNTE), unconjugated peptides and nontreated controls by ~35%. This suggests an enhanced intracellular delivery of PKCβII-. Studies have also shown that SO release is attenuated via trans-activator of transcription-conjugated (Tat; YGRKKRRQRRR) NOX-2 inhibitor (Nox2ds-Tat) (80 μM) by ~37%. There have not been evaluations of Tat- or dual myr-Tat-conjugated PKCβII-. We hypothesize that dual myr-Tat-conjugation would enhance the intracellular delivery of PKCβII- and reduce SO release compared to myr-, Tat-, and myr-Tat-PKCβII- scram. Methods: This study focused on the effects of myr-Tat-PKCβII- on intracellular delivery compared to myr-PKCβII-, Tat -PKCβII-, and myr-Tat-PKCβII- scram, with 0.5% dimethyl sulfoxide (DMSO) vehicle as the control. Sprague-Dawley (SD) male rats (~400g) were anesthetized with isoflurane and given a 0.5% glycogen i.p. injection (16ml), and 16-18hrs later, PMNs were harvested from the peritoneal cavity and incubated for 15min at 37oC with 5μM myr-Tat-PKCβII-, myr-PKCβII-, Tat-PKCβII-, or myr-Tat-PKCβII- scram. PMN SO release was calculated spectrophotometrically by the change in absorbance at 550 nm over 390s via ferricytochrome c reduction after PMA stimulation (100nM). Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Results: Myr-Tat-PKCβII- (n=20, 0.338±0.024, p&lt;0.05) and Myr-PKCβII- (n=8, 0.342±0.041, p&lt;0.05) displayed significant attenuation of SO release by 28% and 27%, respectively, compared to 0.5% DMSO control (n=81, 0.470±0.013). Tat-PKCβII- (n=5, 0.404±0.049) showed attenuated SO release by 14% when compared to the vehicle control. Myr-Tat-PKCβII- scram (n=3, 0.542±0.081) showed an increase in SO release by 15% when compared to the vehicle control. Cell viability was &gt; 85% in all groups. Conclusion: The results of the study suggest that intracellular delivery of PKCβII- cargo using myr-Tat dual conjugation is enhanced when compared to myr-, Tat-conjugated PKCβII-, and myr-Tat-PKCβII- scram. Future studies will utilize western blot analysis and immunohistochemistry to assess the translocation and activity of PKCβII- when conjugated with myr-Tat, myr-, or Tat- peptides. Funding: This study was funded by the National Heart, Lung &amp; Blood Institute (1R43HL160338) SBIR phase I grant that was awarded to Young Therapeutics, LLC. Philadelphia College of Osteopathic Medicine, the Department of Biomedical Sciences, the Division of Research and the Center for Chronic Diseases of Aging. and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Application of copper (I) selective ligands for PET imaging of reactive oxygen species through metabolic trapping

IntroductionReactive oxygen species (ROS) are attractive targets for clinical PET imaging. In this study, we hypothesized that PET imaging of ROS would be possible by using chelating ligands (L) that form stable complexes with copper (I) but not with copper (II), based on metabolic trapping. Namely, when [64Cu][CuI(L)2]+ is oxidized by ROS, the oxidized complex will release [64Cu]Cu2+. Then, the released [64Cu]Cu2+ will be trapped inside the cell, resulting in PET signal depending on the redox potential of ROS. To examine the potential of this novel molecular design for ROS imaging, we synthesized copper (I) complexes with bicinchoninic acid (BCA) disodium salt and bathocuproinedisulfonic acid (BCS) disodium salt and evaluated their reactivity with several kinds of ROS. In addition, the cellular uptake of [64Cu][CuI(BCS)2]3− and the stability of [64Cu][CuI(BCS)2]3− in a biological condition were also evaluated. Methods[64Cu]Cu2+ was reduced to [64Cu]Cu+ by ascorbic acid and coordinated with BCA and BCS in the acetate buffer to synthesize [64Cu][CuI(BCA)2]3− and [64Cu][CuI(BCS)2]3−. The radiochemical yields were determined by thin-layer chromatography (TLC). After [64Cu][CuI(BCS)2]3− was incubated with hydroxyl radical, lipid peroxide, superoxide, and hydrogen peroxide, the percentage of released [64Cu]Cu2+ from the parent complex was evaluated by TLC. HT-1080 human fibrosarcoma cells were treated with 0.1 % Dimethyl sulfoxide (control), imidazole ketone erastin (IKE), or IKE + ferrostatin-1 (Fer-1). Then, the uptake of [64Cu][CuI(BCS)2]3− to HT-1080 cells in each group was evaluated as %Dose/mg protein. Lastly, [64Cu][CuI(BCS)2]3− was incubated in human plasma, and its intact ratio was determined by TLC. ResultsThe radiochemical yield of [64Cu][CuI(BCS)2]3− (86 ± 1 %) was higher than that of [64Cu][CuI(BCA)2]3− (44 ± 3 %). [64Cu][CuI(BCA)2]3− was unstable and partially decomposed on TLC. After [64Cu][CuI(BCS)2]3− was reacted with hydroxyl radical, lipid peroxide, and superoxide, 67 ± 2 %, 44 ± 13 %, and 22 ± 3 % of total radioactivity was detected as [64Cu]Cu2+, respectively. On the other hand, the reaction with hydrogen peroxide did not significantly increase the ratio of [64Cu]Cu2+ (4 ± 1 %). These results suggest that [64Cu][CuI(BCS)2]3− could be used for detecting high-redox-potential ROS such as hydroxyl radical and lipid peroxide with high selectivity. The cellular uptake values of [64Cu][CuI(BCS)2]3− in the control, IKE, and Fer-1 group were 42 ± 2, 54 ± 2, and 47 ± 5 %Dose/mg protein (n = 3), respectively, suggesting the ROS specific uptake of [64Cu][CuI(BCS)2]3−. On the other hand, the intact ratio after the incubation of [64Cu][CuI(BCS)2]3− in human plasma was 9 ± 5 %. ConclusionPET imaging of ROS would be possible by using a copper (I) selective ligand, based on metabolic trapping. Although improvement of the membrane permeability and the stability of copper (I) complexes is required, the present results pave the way for the development of novel 64Cu-labeled complexes for PET imaging of ROS.

Forced Degradation Products of Olmesartan Medoxomil and their In Vitro Genotoxicity Assessment by Comet Assay using HEK Cells

AbstractThis study designed to characterize major forced degradation products of Olmesartan Medoxomil (OM) and to perform genotoxicity profiling using acid, base, and hydrogen peroxide‐induced stress degradation studies. The isolation of degradation products was conducted on flash chromatography using FlashPure ECOFLEX C18 4 g column with gradient elution and diode array detection‐based fraction collection, and characterized by mass spectrometry. Three impurities (impurity‐1, 2, and 3) were identified with respective m/z values of 447.213, 461.229, and 463.208. The genotoxic potential of impurities was assessed by in silico prediction, and in vitro comet assay using HEK cells. The extent of DNA damage was analyzed using Comet Assay IV imaging software. For impurity‐1, head and tail lengths and their respective total intensities were significantly (p&lt;0.001) larger than those of dimethyl sulfoxide (solvent) control and comparable to positive control. Other parameters such as mean grey level and total area of comet were statistically significant as compared with the solvent and positive controls. Overall, impurity‐1 was a possible genotoxic impurity and a known major degradation product in OM tablet dosage form. In silico prediction also revealed impurity‐1 as toxicity class 3, whilst impurities 2 and 3 were of toxicity class 4.

Anti-inflammatory effect of echinacoside in collagen-induced arthritis via Nrf2/Drp1 pathway.

Oxidative damage plays an important role in the progression of rheumatoid arthritis (RA). Emerging research evidence suggests that natural antioxidants may effectively ameliorate this disease. To investigate the therapeutic effect of echinacoside (ECH) in a collagen-induced arthritis (CIA) mouse model and thus elucidate the underlying molecular mechanism in RA. Collagen-induced arthritis mice were intraperitoneally administered 1% dimethyl sulfoxide (DMSO) (control) or 0.6 mg of ECH every other day for 1 month. Arthritis scores and the number of affected paws were assessed. On day 60, mice were euthanized, synovial tissue specimens were obtained, and serum interleukin (IL)-6 and IL-1â expression levels were measured. Mitochondrial morphologies, reactive oxygen species (ROS) content, expression of dynamin-related protein 1 (Drp1), IL-6, nod-like receptor protein 3 (NLRP3), kelch-like ECH-associated protein 1 (Keap1), and nuclear factor-erythroid-2-related factor 2 (Nrf2) contents in synovium were analyzed and compared between DMSOand ECH-treated CIA mice. Following ECH treatment, mitochondria of CIA-induced mice were found to be elongated, while their arthritis scores, inflammation and the number of affected paws, and the expression levels of Drp1, NLRP3, IL-6, ROS, and Keap1 were all found to be significantly reduced. Conversely, the level of antioxidant factor Nrf2 was found to be elevated. Further, mitochondrial fission was found to be inhibited in synovial tissues. Our findings validate the therapeutic efficacy of ECH in the CIA mouse model. Echinacoside may suppress oxidative stress and inhibit inflammation by regulating the Nrf2/Drp1 pathway, thus supporting its utility in the treatment of RA.

Histone Lysine Demethylase KDM5 Inhibitor CPI-455 Induces Astrocytogenesis in Neural Stem Cells.

Lysine-specific histone demethylase 5A (KDM5A) is known to facilitate proliferation in cancer cells and maintain stemness to repress the astrocytic differentiation of neural stem cells (NSCs). In the study presented here, we investigated the effect of a KDM5 inhibitor, CPI-455, on NSC fate control. CPI-455 induced astrocytogenesis in NSCs during differentiation. Kdm5a, but not Kdm5c, knockdown induced glial fibrillary acidic protein (Gfap) transcription. CPI-455 induced signal transducer and activator of transcription 3, increased bone morphogenetic protein 2 expression, and enhanced mothers against decapentaplegic homolog 1/5/9 phosphorylation. The treatment of CPI-455 enhanced the methylation of histone H3 lysine 4 in the Gfap promoter when compared to that of the dimethyl sulfoxide control. In addition, CPI-455 treatment significantly reduced the recruitment of KDM5A to the Gfap promoter. Our data suggest that the KDM5 inhibitor CPI-455 effectively controls NSC cell fate via KDM5A inhibition and induces astrocytogenesis.

Efficacy of Glycicumarin and Isoliquiritigenin in Suppressing Colonic Peristalsis in Both an Animal Model and a Clinical Trial.

Patients with diarrhea-predominant irritable bowel syndrome (IBS-D) show excessive peristalsis, and antispasmodic agents may be useful therapeutic agents. There are few reports on the use of Kampo medicines for the treatment of IBS-D. Shakuyakukanzoto (SKT) is a Kampo medicine that is effective against abdominal pain. We examined the relationship between SKT and intestinal peristalsis in an animal model and a prospective study. In the animal model, SKT and its components were administered from the serosal side of the colon and colonic peristalsis was evaluated using intraluminal pressure and spatiotemporal mapping before and after the administration of SKT and its components. In this clinical trial, we used abdominal ultrasonography (US) to obtain long-axis images of the sigmoid colon of 11 patients. The frequency of intestinal peristalsis was measured using US in five patients with SKT and six patients without medication after the ingestion of a test meal. The primary outcome was the frequency of peristalsis. The Clinical Trial Registry Website (Trial No. UMIN-CTR; UMIN000051547). In the animal model, peony did not suppress peristalsis frequency, but SKT (p = 0.005) and glycyrrhiza (p = 0.001) significantly suppressed peristalsis frequency compared with saline and peony. Among the glycyrrhiza components, glycycoumarin and isoliquiritigenin suppressed the peristalsis frequency compared to dimethyl sulfoxide (control) (p = 0.001, 0.01, respectively). In a clinical trial, peristalsis was significantly suppressed after oral administration in patients taking SKT (p = 0.03). Administration of SKT was found to inhibit colonic peristalsis, with glycicumarin and isoliquiritigenin being particularly relevant among its components.

Galanin receptors modulate cutaneous vasodilation elicited by whole-body and local heating but not thermal sweating in young adults

Galanin receptor subtypes GAL1, GAL2, and GAL3 are involved in several biological functions. We hypothesized that 1) GAL3 receptor activation contributes to sweating but limits cutaneous vasodilation induced by whole-body and local heating without a contribution of GAL2; and 2) GAL1 receptor activation attenuates both sweating and cutaneous vasodilation during whole-body heating. Young adults underwent whole-body (n = 12, 6 females) and local (n = 10, 4 females) heating. Forearm sweat rate (ventilated capsule) and cutaneous vascular conductance (CVC; ratio of laser-Doppler blood flow to mean arterial pressure) were assessed during whole-body heating (water-perfusion suit circulated with warm (35 °C) water), while CVC was also assessed by local forearm heating (from 33 °C to 39 °C and elevated to 42 °C thereafter; each level of heating maintained for ∼30 min). Sweat rate and CVC were evaluated at four intradermal microdialysis forearm sites treated with either 1) 5% dimethyl sulfoxide (control), 2) M40, a non-selective GAL1 and GAL2 receptor antagonist, 3) M871 to selectively antagonize GAL2 receptor, or 4) SNAP398299 to selectively antagonize GAL3 receptor. Sweating was not modulated by any GAL receptor antagonist (P > 0.169), whereas only M40 reduced CVC (P ≤ 0.003) relative to control during whole-body heating. Relative to control, SNAP398299 augmented the initial and sustained increase in CVC during local heating to 39 °C, and the transient increase at 42 °C (P ≤ 0.028). We confirmed that while none of the galanin receptors modulate sweating during whole-body heating, GAL1 receptors mediate cutaneous vasodilation. Further, GAL3 receptors blunt cutaneous vasodilation during local heating.

1641-P: C-C Motif Chemokine Ligand 2 Promotes Myogenesis Recovery, Muscle Strength, and Muscle Performance In Vivo and Has Clinical Relevance for Muscle Performance in Human

Muscle mass decreases with aging, while the C-C motif chemokine ligand 2 (CCL2) increases with aging. In our previous study, CCL2 positively affects in vitro myogenesis. In this context, CCL2 can be considered a potential aging-promoting factor. To further confirm the relationship between muscle and CCL2, we sought to establish the role of CCL2 in vivo and in humans. It was divided into Dimethyl Sulfoxide (DMSO) control, dexamethasone (Dexa) intraperitoneal treatment group, and Dexa with the CCL2 treatment group. As a result, grip power and exercise endurance were reduced in the Dexa treatment group compared to the control group but recovered with the CCL2 treatment. In muscle mass, there was no significant difference in mass between groups. We examined the cross-sectional area distribution (CSA) with the tibialis anterior muscle. The Dexa treatment group was distributed between CSA 1000-3000 µm² and was left-shifted. However, the Dexa and CCL2 group was similar or slightly higher than the DMSO group; it was right-shifted and distributed in the 2000-5000 µm². In humans, the median value was 303.8 pg/ml in the no sarcopenia group and 321.7 pg/ml in the sarcopenia group. Subsequently, the CCL2 level was checked by dividing the groups into whether there was sarcopenia, low mass, low strength, or low speed. The CCL2 level was statistically significantly higher in the group showing low speed. In conclusion, CCL2 promotes myogenesis recovery, muscle strength, and muscle performance in vivo and has clinical relevance for muscle performance in humans. Disclosure E.Hong: None. E.Ha: None. J.Lee: None. Y.Choi: None. K.Lee: None. J.Won: None. M.Kwak: None. Funding Ministry of Education, Republic of Korea (2020R1I1A3073230)

Nifuroxazide induces the apoptosis of human non‑small cell lung cancer cells through the endoplasmic reticulum stress PERK signaling pathway.

The aim of the present study was to investigate the molecular mechanism of nifuroxazide (NFZ) in the induction of apoptosis of NCI-H1299 human non-small cell lung cancer (NSCLC) cells through the reactive oxygen species (ROS)/Ca2+/protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-DNA damage inducible transcript 3 (CHOP) signaling pathway. Morphological changes of cells were observed by microscopy, and the apoptosis and intracellular ROS levels of cells were observed by inverted fluorescence microscopy. Cell viability after the addition of the PERK inhibitor, GSK2606414, were detected by Cell Counting Kit-8 assay. Annexin V-FITC was used to detect cell apoptosis, Brite 670 was used to detect intracellular ROS and Fura Red AM was used to detect Ca2+ content. Western blotting was used to detect PERK, phosphorylated (P)-PERK, ATF4, CHOP, P-Janus kinase 2 and P-signal transducer and activator of transcription 3 expression levels. Compared with the dimethyl sulfoxide control group, NFZ inhibited the survival activity in the H1299 NSCLC cell line, in a time- and dose-dependent manner. However, GSK2606414 inhibited the NFZ-induced apoptosis of H1299 cells. GSK2606414 also inhibited the increase in ROS and Ca2+ in H1299 cells induced by NFZ. Western blotting results demonstrated that NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP, whereas GSK2606414 significantly reduced the NFZ-induced increase in these protein expression levels. In conclusion, NFZ may induce the apoptosis of H1299 NSCLC cells through the ROS/Ca2+/PERK-ATF4-CHOP signaling pathway.

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis

Introduction: Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology. Methods: Zebrafish larvae with a developed visual system at three days post-fertilization (dpf) were exposed to CSE for 4, 24 or 48 hours. Visual behaviour, hyaloid vasculature morphology, retinal histology, oxidative stress gene expression and outer segment phagocytosis were investigated using visual behavioural optokinetic and visual motor response assays (OKR and VMR), microscopy (light, fluorescence and transmission electron microscopy), and real-time PCR. Results: In zebrafish larvae, 48 hours of CSE treatment resulted in significantly reduced visual behaviour. Larvae treated with 10, 15 or 20 μg/mL CSE showed an average of 13.7, 10.7 or 9.4 saccades per minute, respectively, significantly lower compared with 0.05% DMSO controls (p=0.0093, p=0.0004 and p&lt;0.0001, respectively) that exhibited 19.7 saccades per minute. The diameter of intraocular vessels increased from 4.12 μm in 0.05% DMSO controls to 5.69 μm in the 20 μg/mL CSE-treated larvae (p≤0.0001). Biometry analysis highlighted a significant axial length elongation in 20 μg/mL CSE-treated larvae (216.9 μm, p&lt;0.0001) compared to 0.05% dimethyl sulfoxide (DMSO) controls (205.1 μm). Larvae exposed to 20 μg/mL CSE had significantly (p=0.0002) higher numbers of RPE phagosomes compared to vehicle controls (0.1425 and 0.093 phagosomes/μm RPE, respectively). Conclusions: Zebrafish larvae with a developed visual system display apparent defects in visual behaviour and retinal biology after acute exposure to CSE, establishing a valuable in vivo model to investigate ocular disorders related to cigarette smoke.

Genotoxic effects of particulate matter on larvae of a common and widespread butterfly along an urbanization gradient

Biodiversity is currently declining worldwide. Several threats have been identified such as habitat loss and climate change. It is unknown if and how air pollution can work in addition or in synergy to these threats, contributing to the decline of current species and/or local extinction. Few studies have investigated the effects of particulate matter (PM), the main component of air pollution, on insects, and no studies have investigated its genotoxic effects through Micronucleus assay. Butterflies play an important role in the environment, as herbivores during larval stages, and as pollinators as adults. The aim of this study was to evaluate the genotoxic effects of PM10 from different sites along a gradient of population urbanization, on a common cabbage butterfly species (Pieris brassicae). PM10 was collected from April to September in an urban (Turin, Italy), a suburban (Druento, Italy) and a mountain site (Ceresole Reale, Italy) with different urbanization levels. P. brassicae larvae (n = 218) were reared in the laboratory under controlled conditions (26 °C, L:D 15:9) on cabbage plants (average 9.2 days), and they were exposed to PM10 organic extracts (20 and 40 m3/mL) or dimethyl sulfoxide (controls) through vaporization. After exposure, larvae were dissected and cells were used for the Micronucleus (MN) assay. Results showed that all PM extracts induced significant DNA damage in exposed larvae compared to controls, and that increasing the PM dose (from 20 to 40 m3/mL) increased genotoxic effects. However, we did not detect any significant differences between sites with different urbanization levels. In conclusion, PM at different concentrations induced genotoxic effects on larvae of a common butterfly species. More alarmingly, PM could work in addition to and/or in synergy with other compounds (e.g. pesticides) and, especially on species already threatened by other factors (e.g. fragmentation), thus affecting the vitality of populations, leading to local extinctions.

Impact of the Voltage-Gated Calcium Channel Antagonist Nimodipine on the Development of Oligodendrocyte Precursor Cells

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

Cabbage butterfly as bioindicator species to investigate the genotoxic effects of PM10.

Atmospheric pollution poses a serious threat to environment and human health, and particulate matter (PM) is one of the major contributors. Biological effects induced by PM are investigated through in vitro assays using cells and by in vivo tests with laboratory model animals. However, also the estimation of adverse effects of pollutants, including airborne ones, on wild animals, such as insects, is an essential component of environmental risk assessment. Among insects, butterflies are sensitive to environmental changes and are important wild pollinators, so they might be suitable as environmental bioindicator species. The aim of this study was to evaluate the suitability of a wild cabbage butterfly species (Pieris brassicae) as a bioindicator organism to assess the genotoxic effects of PM10 collected in different sites. PM10 was collected from April to September in urban, suburban, and rural sites. P. brassicae larvae were reared in laboratory under controlled conditions on cabbage plants and exposed to PM10 organic extracts or dimethyl sulfoxide (controls) through vaporization. After exposure, larvae were dissected, and cells were used for comet assay. All PM extracts induced significant DNA damage in exposed larvae compared to controls and the extract collected in the most polluted site caused the highest genotoxic effect. In conclusion, the study suggested that butterflies, such as P. brassicae, could be applied as sensitive and promising bioindicators to investigate air quality and PM genotoxicity. Indeed, the use of these organisms allows the detection of genotoxic effects induced by PM sampled also in low-polluted areas.

In vitro adverse effects of amitraz on semen quality: Consequences in bovine embryo development

Veterinary drugs are potential environmental pollutants that interfere with male reproductive function. Infertility has increased, and it is known that environmental toxins contribute to declining sperm parameters. Amitraz {N,N-[(methylamino) dimeth-ylidyne] di-2,4-xylidine} (AMZ) is a formamidine pesticide widely used as an insecticide and an acaricide. The aim of this study was to evaluate the toxicity of AMZ in bovine sperm. Three experiments using frozen-thawed bovine semen incubated with AMZ for 2 h were carried out. Negative and solvent (dimethyl sulfoxide) controls were run simultaneously with treatments. In experiment 1, the AMZ concentrations used were 10, 15 and 25 μg AMZ/ml and the sperm parameters evaluated were viability, mitochondrial activity, acrosomal status, functional membrane integrity and apoptosis. In experiments 2 and 3, 25 μg AMZ/ml was used to evaluate fertilizing capacity, embryo development and blastocyst DNA damage. In experiment 1, 25 μg AMZ/ml decreased sperm viability (P = 0.01), reduced mitochondrial activity (P = 0.03) and induced apoptosis (P < 0.01). Also, 15 and 25 μg AMZ/ml affected functional membrane integrity (P < 0.01). In experiment 2, AMZ did not alter sperm-zona binding (P = 0.40) and pronucleus formation (P = 0.36). In experiment 3, 25 μg AMZ/ml decreased the rate of embryo development (P < 0.01) and increased apoptosis (P = 0.03). These results suggest that AMZ induced alterations in bovine sperm, probably affecting male fertility at concentrations that could be present in the environment.

Retinoic acid as a teratogen: IX-Induction of fetal skeletal anomalies and alteration in the utero-placental expression pattern of EGFR during mice development

ABSTRACT Retinoic acid (RA), an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Nevertheless, it has long been recognized that overexposure to vitamin A or retinoic acid causes widespread teratogenesis in rodents as well as humans. However, the mechanism of its teratogenic effect is not yet understood. Here, we examined the effects of RA on the fetal and placental development as an attempt to find the mechanism of its teratogenicity through inhibition of utero-placental development. The pregnant mice were divided into three groups: RA treated group, control treated with dimethyl sulfoxide (DMSO) group and control non-treated group. The RA treated group received 15 mg/kg RA by intraperitoneal injection from 8.5 to 11.5 gestation days. The pregnant mice were sacrificed by cervical dislocation on days 9.5, 10.5, 12.5, 13.5 and 18.5 of gestation. The uteri were isolated, and the fetuses have been exposed to morphological examination and skeletal staining. Moreover, histological and immunohistochemical studies on placenta have been done. Morphologically, we recorded a reduction in the number of implanted fetuses besides hematoma in different parts of the uteri of the RA treated animals. Furthermore, RA was found to induce decreases in fetal weight and length, as well as malformations in the tail, forelimbs and hindlimbs, which were confirmed by skeletal examination. Interestingly, RA treatment showed a decrease in the expression level of epidermal growth factor receptor (EGFR) during placenta development, especially in junctional and labyrinth zones. We concluded that high dose of RA may have a direct teratogenic effect on the fetus besides an indirect effect through modulating EGFR signal during the placenta development.

The Modulatory Role of Orexin 1 Receptor in Nucleus Accumbens (NAc) on Spatial Memory in Rats.

Neuropeptide orexin mainly exists in neurons within and around the lateral hypothalamus and exhibits high affinity to orexin 1 and 2 receptors (OX1R and OX2R, respectively). Orexinergic neurons send their axons to the nucleus accumbens (NAc), which expresses OX1Rs. Previous studies have shown the involvement of orexins and their role in learning and memory processes in the dorsal raphe nucleus and hippocampus. However, no study has examined the effects of orexins in the NAc on memory. The present study examined the effect of the post-training and pre-probe trial intra-NAc administration of SB-33486-A (OX1R antagonist, 12 μg/0.5μl) and TCS-OX2-29 (OX2R antagonist, 10 μg/0.5 μl) on the consolidation and retrieval of memory in the Morris Water Maze (MWM) task. In experiment 1, rats were trained in the MWM and, immediately after every training, received bilateral injections of dimethyl sulfoxide (DMSO) (control group), SB-334867-A (SB), and TCS-OX2-29 (TCS) into the NAc. Experiment 2 was similar to experiment 1, except that the rats received DMSO, SB, and TCS 15 min before the probe test. Probe and visible tests were performed after the last training, and the distance moved, escape latency, and velocity were recorded. In experiment 3, rats trained in experiments 1 and 2, immediately after the probe test, were given the trials for visuomotor coordination assessment on the visible platform. According to the results, the injection of SB increased the distance moved and escape latency in the treated groups, compared to the control group, in the consolidation phase of spatial memory (P<0.05) but not in its retrieval phase (P>0.05). However, TCS-OX2-29 had no effect. These results suggest that the inactivation of the NAc OX1Rs, but not OX2Rs, impairs the consolidation but not the retrieval of spatial memory in rats.