Fentanyl Overdose Causes Prolonged Cardiopulmonary Dysregulation in Male SKH1 Mice.

Fentanyl overdose is a survivable condition that commonly resolves without chronic overt changes in phenotype. While the acute physiological effects of fentanyl overdose, such as opioid-induced respiratory depression (OIRD) and Wooden Chest Syndrome, represent immediate risks of lethality, little is known about longer-term systemic or organ-level impacts for survivors. In this study, we investigated the effects of a single, bolus fentanyl overdose on components of the cardiopulmonary system up to one week post. SKH1 mice were administered subcutaneous fentanyl at the highest non-lethal dose (62 mg/kg), LD10 (110 mg/kg), or LD50 (135 mg/kg), before euthanasia at 40 min, 6 h, 24 h, or 7 d post-exposure. The cerebral cortex, heart, lungs, and plasma were assayed using an immune monitoring 48-plex panel. The results showed significantly dysregulated cytokine, chemokine, and growth factor concentrations compared to time-matched controls, principally in hearts, then lungs and plasma to a lesser extent, for the length of the study, with the cortex largely unaffected. Major significant analytes contributing to variance included eotaxin-1, IL-33, and betacellulin, which were generally downregulated across time. The results of this study suggest that cardiopulmonary toxicity may persist from a single fentanyl overdose and have wide implications for the endurance of the expanding population of survivors.

Mitochondrial sequencing identifies long noncoding RNA features that promote binding to PNPase.

Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.

Fentanyl overdose: Temporal effects and prognostic factors in SKH1 mice.

Fentanyl exposure and overdose are growing concerns in public health and occupational safety. This study aimed to establish parameters of fentanyl lethality in SKH1 mice for future overdose research. Lethality was determined using the up-down procedure, with subjects monitored post-administration using pulse oximetry (5 min) and then whole-body plethysmography (40 min). Following the determination of subcutaneous dose-response, [18F]Fluorodeoxyglucose positron emission tomography (18 F-FDG PET) was performed after LD10 fentanyl at 40 min, 6 h, 24 h or 7 days post-dose. LD10 and LD50 were observed to be 110 and 135 mg/kg, respectively, and consistent with four-parameter logistic fit values of 111.2 and 134.6 mg/kg (r2  = 0.9996). Overdose (LD10 or greater) yielded three distinct cardiovascular groups: survival, non-survival with blood oxygen saturation (SpO2) minimum ≥37% and non-survival with SpO2 <37%. Breaths per minute, minute volume and inspiratory quotient were significantly different between surviving and non-surviving animals for up to 40 min post-injection. 18 F-FDG PET revealed decreased glucose uptake in the heart, lungs and brain for up to 24 h. These findings provide critical insights into fentanyl lethality in SKH1 mice, including non-invasive respiratory effects and organ-specific impacts that are invaluable for future translational studies investigating the temporal effects of fentanyl overdose.

Opioids and Vitamin C: Known Interactions and Potential for Redox-Signaling Crosstalk.

Opioids are among the most widely used classes of pharmacologically active compounds both clinically and recreationally. Beyond their analgesic efficacy via µ opioid receptor (MOR) agonism, a prominent side effect is central respiratory depression, leading to systemic hypoxia and free radical generation. Vitamin C (ascorbic acid; AA) is an essential antioxidant vitamin and is involved in the recycling of redox cofactors associated with inflammation. While AA has been shown to reduce some of the negative side effects of opioids, the underlying mechanisms have not been explored. The present review seeks to provide a signaling framework under which MOR activation and AA may interact. AA can directly quench reactive oxygen and nitrogen species induced by opioids, yet this activity alone does not sufficiently describe observations. Downstream of MOR activation, confounding effects from AA with STAT3, HIF1α, and NF-κB have the potential to block production of antioxidant proteins such as nitric oxide synthase and superoxide dismutase. Further mechanistic research is necessary to understand the underlying signaling crosstalk of MOR activation and AA in the amelioration of the negative, potentially fatal side effects of opioids.

Contribution of HCN1 variant to sinus bradycardia: A case report.

BACKGROUND: Missense mutations in the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia. OBJECTIVE: To investigate possible HCN4 channel mutation in a young patient with profound sinus bradycardia. METHODS: Direct sequencing of HCN4 and whole-exome sequencing were performed on DNA samples from the indexed patient (P), the patient's son (PS), and a family unrelated healthy long-distance running volunteer (V). Resting heart rate was 31 bpm for P, 67 bpm for PS, and 50 bpm for V. Immunoblots, flow cytometry, and immunocytofluorescence confocal imaging were used to study cellular distribution of channel variants. Patch-clamp electrophysiology was used to investigate the properties of mutant HCN1 channels. RESULTS: In P no missense mutations were found in the HCN4 gene; instead, we found two heterozygous variants in the HCN1 gene: deletion of an N-terminal glycine triplet (72GGG74, "N-del") and a novel missense variant, P851A, in the C-terminal region. N-del variant was found before and shared by PS. These two variations were not found in V. Compared to wild type, N-del and P851A reduced cell surface expression and negatively shifted voltage-activation with slower activation kinetics. CONCLUSION: Decreased channel activity HCN1 mutant channel makes it unable to contribute to early depolarization of sinus node action potential, thus likely a main cause of the profound sinus bradycardia in this patient.

Corticosterone and chlorpyrifos oxon exposure elicits spatiotemporal MAPK phosphoprotein signaling in a mouse brain.

Chlorpyrifos (CPF) is one of the most widely-used pesticides globally for agricultural purposes. Certain occupations (e.g., farmers, military) are at an increased risk for high-dose exposure to CPF, which can lead to seizures and irreversible brain injury. Workers with the highest risk of exposure typically experience increased circulating cortisol levels, which is related to physiological stress. To better represent this exposure scenario, a mouse model utilized exogenous administration of corticosterone (CORT; high physiologic stress mimic) in combination with chlorpyrifos oxon (CPO; oxon metabolite of CPF); this combination increases neuroinflammation post-exposure. In the present study adult male C57BL/6J mice were given CORT (200 µg/mL) in drinking water for seven days followed by a single intraperitoneal injection of CPO (8.0 mg/kg) on day eight, and euthanized 0.5, 2, and 24 h post-injection. Ten post-translationally modified proteins were measured in the frontal cortex and striatum to evaluate brain region-specific effects. The spatiotemporal response to CORT + CPO sequentially activated phosphoproteins (p-ERK1/2, p-MEK1/2, p-JNK) involved in mitogen-activated protein kinase (MAPK) signaling. Observed p-ZAP70 responses further integrated MAPK signaling and provided a spatiotemporal connection between protein phosphorylation and neuroinflammation. This study provides insight into the spatiotemporal cellular signaling cascade following CORT + CPO exposure that represent these vulnerable populations.

Differential phosphoprotein signaling in the cortex in mouse models of Gulf War Illness using corticosterone and acetylcholinesterase inhibitors.

AIMS: Veterans from the 1990-91 Gulf War were exposed to acetylcholinesterase inhibitors (AChEIs), and, following service, an estimated one-third began suffering from a medically unexplained, multi-symptom illness termed Gulf War Illness (GWI). Previous research has developed validated rodent models that include exposure to exogenous corticosterone (CORT) and AChEIs to simulate high stress and chemical exposures encountered in theater. This combination of exposures in mice resulted in a marked increase in neuroinflammation, which is a common symptom of veterans suffering from GWI. To further elucidate the mechanisms associated with these mouse models of GWI, an investigation into intracellular responses in the cortex were performed to characterize the early cellular signaling changes associated with this exposure-initiated neuroinflammation. MAIN METHODS: Adult male C57BL/6J mice were exposed to CORT in the drinking water (200 µg/mL) for 7 days followed by a single intraperitoneal injection of diisopropyl fluorophosphate (DFP; 4.0 mg/kg) or chlorpyrifos oxon (CPO; 8.0 mg/kg), on day 8 and euthanized 0.5, 2, and 24 h post-injection. Eleven post-translationally modified protein targets were measured using a multiplexed ELISA. KEY FINDINGS: Phosphoprotein responses were found to be exposure specific following AChEI insult, with and without CORT. Specifically, CORT + CPO exposure was found to sequentially activate several phosphoproteins involved in mitogen activated protein kinase signaling (p-MEK1/2, p-ERK1/2, and p-JNK). DFP alone similarly increased proteins in this pathway (p-RPS6, and p-JNK), but the addition of CORT ameliorated these affects. SIGNIFICANCE: The results of this study provide insight into differentially activated pathways depending on AChEI in these GWI models.

Opening large-conductance potassium channels selectively induced cell death of triple-negative breast cancer.

BACKGROUND: Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC. METHODS: Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice. RESULTS: In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity. CONCLUSIONS: Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.

Comparative analysis of lung and blood transcriptomes in mice exposed to multi-walled carbon nanotubes.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNT) causes inflammation, fibroproliferation, immunotoxicity, and systemic responses in rodents. However, the search for representative biomarkers of exposure is an ongoing endeavor. Whole blood gene expression profiling is a promising new approach for the identification of novel disease biomarkers. We asked if the whole blood transcriptome reflects pathology-specific changes in lung gene expression caused by MWCNT. To answer this question, we performed mRNA sequencing analysis of the whole blood and lung in mice administered MWCNT or vehicle solution via pharyngeal aspiration and sacrificed 56 days later. The pattern of lung mRNA expression as determined using Ingenuity Pathway Analysis (IPA) was indicative of continued inflammation, immune cell trafficking, phagocytosis, and adaptive immune responses. Simultaneously, innate immunity-related transcripts (Plunc, Bpifb1, Reg3g) and cancer-related pathways were downregulated. IPA analysis of the differentially expressed genes in the whole blood suggested increased hematopoiesis, predicted activation of cancer/tumor development pathways, and atopy. There were several common upregulated genes between whole blood and lungs, important for adaptive immune responses: Cxcr1, Cd72, Sharpin, and Slc11a1. Trim24, important for TH2 cell effector function, was downregulated in both datasets. Hla-dqa1 mRNA was upregulated in the lungs and downregulated in the blood, as was Lilrb4, which controls the reactivity of immune response. "Cancer" disease category had opposing activation status in the two datasets, while the only commonality was "Hypersensitivity". Transcriptome changes occurring in the lungs did not produce a completely replicable pattern in whole blood; however, specific systemic responses may be shared between transcriptomic profiles.

Chronic, Recreational Chloroform-Induced Liver Injury.

Historically used as an anesthetic, chloroform is a halogenated hydrocarbon that is associated with central nervous system depression, arrhythmias, and hepatotoxicity. It is no longer used clinically, but accidental and intentional poisonings still occur. We report a case of chronic chloroform abuse leading to severe hepatotoxicity in a 26-year-old male graduate student. The patient presented to the emergency department with a three-day history of abdominal pain, dehydration, and scleral icterus. He drank several beers the night before the onset of symptoms, but denied taking acetaminophen, ibuprofen, or other drugs. An extensive work-up revealed an aspartate aminotransferase (AST) of 13,527 U/L and alanine aminotransferase (ALT) of 8,745 U/L, but the cause of his liver injury could not be determined. It was not until many months later that the patient admitted to inhaling chloroform in the weeks leading up to his illness.

Transcriptome profiling reveals novel BMI- and sex-specific gene expression signatures for human cardiac hypertrophy.

How obesity or sex may affect the gene expression profiles of human cardiac hypertrophy is unknown. We hypothesized that body-mass index (BMI) and sex can affect gene expression profiles of cardiac hypertrophy. Human heart tissues were grouped according to sex (male, female), BMI (lean<25 kg/m2, obese>30 kg/m2), or left ventricular hypertrophy (LVH) and non-LVH nonfailed controls (NF). We identified 24 differentially expressed (DE) genes comparing female with male samples. In obese subgroup, there were 236 DE genes comparing LVH with NF; in lean subgroup, there were seven DE genes comparing LVH with NF. In female subgroup, we identified 1,320 significant genes comparing LVH with NF; in male subgroup, there were 1,383 significant genes comparing LVH with NF. There were seven significant genes comparing obese LVH with lean NF; comparing male obese LVH with male lean NF samples we found 106 significant genes; comparing female obese LVH with male lean NF, we found no significant genes. Using absolute value of log2 fold-change > 2 or extremely small P value (10-20) as a criterion, we identified nine significant genes (HBA1, HBB, HIST1H2AC, GSTT1, MYL7, NPPA, NPPB, PDK4, PLA2G2A) in LVH, also found in published data set for ischemic and dilated cardiomyopathy in heart failure. We identified a potential gene expression signature that distinguishes between patients with high BMI or between men and women with cardiac hypertrophy. Expression of established biomarkers atrial natriuretic peptide A (NPPA) and B (NPPB) were already significantly increased in hypertrophy compared with controls.

Altering calcium influx for selective destruction of breast tumor.

BACKGROUND: Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. METHODS: We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. RESULTS: Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. CONCLUSIONS: We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Altering bioelectricity on inhibition of human breast cancer cells.

BACKGROUND: Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer. METHODS: In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive). RESULTS: Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10-20 µM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 µM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil. CONCLUSIONS: Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

The preliminary study of effects of tolfenamic Acid on cell proliferation, cell apoptosis, and intracellular collagen deposition in keloid fibroblasts in vitro.

Keloid scarring is a fibroproliferative disorder due to the accumulation of collagen type I. Tolfenamic acid (TA), a nonsteroidal anti-inflammatory drug, has been found to potentially affect the synthesis of collagen in rats. In this preliminary study, we aimed to test the effects of TA on cell proliferation, cell apoptosis, and the deposition of intracellular collagen in keloid fibroblasts. Normal fibroblasts (NFs) and keloid fibroblasts (KFs) were obtained from human dermis tissue. Within the dose range 10(-3)-10(-6) M and exposure times 24 h, 48 h, and 72 h, we found that 0.55 × 10(-3) M TA at 48 h exposure exhibited significantly decreased cell proliferation in both NFs and KFs. Under these experimental conditions, we demonstrated that (1) TA treatment induced a remarkable apoptotic rate in KFs compared to NFs; (2) TA treatment reduced collagen production in KFs versus NFs; (3) TA treatment decreased collagen type I expression in KFs comparing to that of NFs. In summary, our data suggest that TA decreases cell proliferation, induces cell apoptosis, and inhibits collagen accumulation in KFs.

Toe Necrosis, Etiologies and Management, a Case Series.

Toe necrosis may have vast different etiologies. These include ischemia, embolus, and others. (1) The most common etiology is ischemia. It is a reduction in blood supply to a viable tissue that can lead to susceptibility to infection and tissue death. Peripheral ischemia, which is rooted in the lower limbs, is a major risk factor for toe necrosis because the basal metabolic requirements of tissue are not being sufficiently met. As a result, pain, ulcers, and gangrene commonly occur. (2) Other causes of direct and indirect toe necrosis and related lower limb gangrene include mechanical trauma, infectious, pharmacological sensitivity, cancer, blue toe syndrome, and other granulomatous diseases, such as Churg-Strauss syndrome. We present a case series of toes necrosis which resulted from different etiologies and their management.

Role of Hyaluronic Acid Treatment in the Prevention of Keloid Scarring.

BACKGROUND: Keloids are benign dermal scars characterized by enhanced growth factor signaling, hyperproliferation activity and reduced extracellular matrix (ECM) deposition of hyaluronic acid. Our hypothesis is that high molecular weight HA can be used to replenish HA deposition in keloids thereby normalizing the keloid fibroblast phenotype. METHODS: One normal (NF1) fibroblast culture and five keloid (KF1, KF2, KF3, KF4, KF5) fibroblast cultures were analyzed for changes in hyperproliferation, growth factor production and extracellular matrix deposition following 72 hour treatment with or without 10 µg/ml HA. RESULTS: Proliferation activity decreased significantly in KF3 following HA treatment. Pro-collagen I expression in KF2 was decreased following HA treatment in association with changes in fiber arrangement to more parallel collagen bundles. In addition, HA demonstrated a downregulation on TGF-b1 growth factor expression in KF3 and KF4 and a decrease in active TGF-b1 release in KF2 and KF5 using ELISA. CONCLUSION: Our data demonstrates that HA has the potential to normalize keloid fibroblast characteristic features such as hyperproliferation, growth factor production and ECM deposition depending on the specific genotype of the keloid fibroblast cell line. This study suggests that high molecular weight HA can be used to replenish HA deposition in keloid fibroblasts thereby decreasing fibrosis and ultimately decreasing keloid manifestation.

Advanced Skin Care - A Novel Ingredient.

The skin provides the human body with protection and a major barrier to environmental assault. Caring for skin is sometimes an afterthought. In other words, if something isn't broken, don't fix it. However, in the case of the integument, nothing could be further from the truth. Intact skin is paramount to health and well-being. This article will review skin care, specifically, advanced skin care, uncovering novel ingredients, and their importance for prevention and treatment as well as delving into the caring for the skin from the outside in.