Fatal Case of Diazepam and Paraquat Poisoning – A Case Report

Background: A very dangerous activity among youth and young adults is the indiscriminate mixing and sharing of prescription drugs, often in combination with alcohol or other drugs. The effects of these combinations of substances can be fatal.Case Report: A 28 years old adult male with alleged history of diazepam and paraquat poisoning was admitted with complaints of chest discomfort, epigastric pain, vomiting and drowsiness. The patient developed acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS) and expired on the next day. To conclude, diazepam even though considered to be a safer drug, has risk of drug abuse and is fatal when taken in overdose along with other central nervous system depressants. Paraquat is a highly toxic compound widely used as herbicide and ingestion of the drug causes death due to respiratory failure. Conclusion: The present study emphasizes on the proper surveillance of diazepam intake in known psychiatric patients and strict rules must be enforced by the Government on marketing of herbicides and pesticides

A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications

ApoE Isoforms Inhibit Amyloid Aggregation of Proinflammatory Protein S100A9

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer’s disease (AD). The amyloid co-aggregation of S100A9 with amyloid-β (Aβ) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD

Protective effect of HDL on NADPH oxidase-derived super oxide anion mediates hypoxia-induced cardiomyocyte apoptosis.

Cardiovascular diseases are the leading cause of death of death in Taiwan. Atherosclerosis can lead to serious problems, including heart attack, stroke, or even death. Coronary heart disease (CHD) occurs when plaque builds up in the coronary arteries to cause the ischemic heart disease which will enhance myocardial remodeling and also induce myocardial hypoxia. High density lipoprotein (HDL) has been proposed to have cardio-protective effects. Under hypoxic conditions (1%O2 for 24hr), in H9c2 cells, reactive oxygen species (ROS) is induced which leads to cardiomyocyte apoptosis and cardiac dysfunction. Therefore, the present study described the protective effect of HDL on hypoxia-induced cardiomyocyte damage. We investigated the NADPH oxidase-produced ROS-related signaling pathways and apoptosis in cardiomyocytes under hypoxia conditions. Results showed that the ROS mediated cardiac damage might occur via AT1 and PKC activation. Furthermore, hypoxia downregulated the survival protein (p-AKTser473) and anti-apoptotic protein (BCL2), whereas pro-apoptotic protein, Bax and caspase 3 were upregulated. These detrimental effects by ROS and apoptosis were prevented by HDL pretreatment. Our findings revealed the underlying molecular mechanism by which HDL suppresses the hypoxia-induced cardiomyocyte dysfunction. Further, we elucidated the role of HDL on preventing hypoxia induced cardiomyocyte apoptosis is mediated through the inhibition of NADPH oxidase-derived ROS

Inhibitory effect of HDL on hypoxia induced ROS production in H9c2 cells.

<p>(A) Representative western blots. ‘Image J’ software was used to calculate the expression level of protein (B). ROS was examined by DCF-AM (10μM), DHE (5μM), and MitoSOX^™ (5μM). Fluorescence intensity of cells was measured by flow cytometry. (C) Neonatal cardiomyocytes were treated with HDL (25–100μg/ml) for 2h, or NAC (500 μM), roteone (5 μM), and then incubated with 1%hypoxia for an additional 24h, and followed by 1h incubation with DHE (10μM). Fluorescence intensity of cells was measured by immunofluorescence microscopy (Olympus CKK53). Data showed the means ± SEM of 3 independent analyses.^#P<0.05 comparison of control and hypoxia groups, *P<0.05 HDL/ NAC treated groups vs. hypoxia treatment.</p

Effect of hypoxia-induced apoptosis and activation of MAPK family proteins, and survival proteins in H9c2 cells.

<p>(A) Survival proteins for 0-24h. (B) MAPK family (p-ERK, p-JNK, p-P38). (C)Fluorescence images show the cells stained with 4, 6-diamidino-2-phenylindole (DAPI) and stained using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay. (D) Graphical representation of TUNEL assay. Data showed the means ± SEM of 3 independent analyses. *P<0.05 vs. hypoxia treatment.</p

Hypoxia-increased oxidative stress in H9c2 cells.

<p>(A) The level of NADPH oxidase (Nox2-gp91 ^phox, p47^phox, and Rac1) protein expressions. (B) The level of AT1 receptor, p-PKCα and p-PKCδ, and antioxidant enzyme SOD2 protein expressions. ‘Image J’ software was used to calculate the expression level of protein.</p

Inhibitory effect of HDL on hypoxia induced MAPK and apoptotic protein expression in H9c2 cells.

<p>(A). Representative western blots. ‘Image J’ software was used to calculate the expression level of protein. (B) Fluorescence images show the cells stained with 4,6-diamidino-2-phenylindole (DAPI) (upper panel) and stained using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay (bottom panel), and photomicrographs were from immunofluorescence microscopy (Olympus CKK53). C). Graphical representation of TUNEL assay. ^#P<0.05 comparison of control and hypoxia groups, *P<0.05 HDL treated groups vs. hypoxia treatment.</p

Schematic representation of the effect of hypoxia and the preventive role of HDL on H9c2 cells.

<p>Hypoxia induces ROS mediated apoptosis through the overexpression of AT1 and PKC, which is prevented by the treatment of HDL.</p