A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

The mechanism of cell death induced by silver nanoparticles is distinct from silver cations

Background Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant. Methods Cells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry. Results We identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts. Conclusions There are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

Effects of Hydroethanolic Extract of Adenia lobata (Jacq.) Engl. (Passifloraceae) on Nociception and Subsequent Anxiety-like Behavior: Both Anti-inflammatory and Antioxidant Approaches

Aims: Adenia lobata (Jacq.) Engl. (Passifloraceae) is widely used in Ivorian traditional pharmacopeia to heal various chronic diseases, relieve headache and pain of gingiva inflammation, and facilitate labor. Here, we investigated the effects of hydroethanolic extract Adenia lobata (HEAL) on nociceptive pain and subsequent anxiety-like behavior. Materials and Methods: We used several experimental pain tests as the writhing, formalin and hot plate to evaluate both antinociceptive and anti-inflammatory actions of the extract. Anxiety related to nociception was tested with open field and elevated plus maze tests. Then, mice were sacrificed for assessing some oxidative stress markers. Results: The extract of 30 mg/kg, p.o. reduced in the similar manner as reference peripheral drug salcylicacetic acid (ASA, 200 mg/kg, i.p.) the number of writhings induced by acid acetic. In both neurogenic and inflammatory phases of formalin test, the extract demonstrated an effective antinociceptive activity than ASA, but comparable to central analgesic tramadol (50 mg/kg, i.p). However, Adenia lobata reduced lesser thermal-induced pain than tramadol in hot plate test, but significantly compared to ASA. Furthermore, HEAL altered anxiety-like behavior in each case of the pain condition studied. Also, the extract showed the highest antioxidant activity by reduction oxide nitric (NO) and malondialdehyde (MDA), and increase non protein thiol (NP-SH) levels. Conclusion: In conclusion, HEAL possesses antinociceptive and anti-inflammatory actions on peripheral and central mechanisms of pain. The phytochemicals components of the extract as alkaloids and flavonoids suggest to interact with the opioid system and combat the oxidative stress, respectively. Our findings provide scientific basis for the use of Adenia lobata in traditional medicine against pain and related diseases.

Human Cystathionine γ-Lyase Is Inhibited by s-Nitrosation: A New Crosstalk Mechanism between NO and H2S

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

RedoxiFluor: A microplate assay to quantify protein thiol redox state in percentages and moles

An accessible, time- and cost-efficient microplate assay to quantify protein thiol redox state in percentages and moles relative to the thiol proteome (i.e., context) and other targets (i.e., array mode) would be invaluable for understanding how protein thiols regulate essential biological processes. RedoxiFluor achieves several key benefits (i.e., percentages, moles, context, array mode) in a microplate format. After robustly validating RedoxiFluor, comparative analysis reveals that key benefits are intractable to other immunological techniques. Moles is an unprecedented achievement. Proof-of-concept studies illuminating fundamental redox principles (i.e., specificity, context, and heterogeneity) through measurement alone demonstrate how RedoxiFluor can advance understanding. For example, target specific protein thiol redox state changes are: (1) context specific (i.e., redox stimulus dependent); (2) selective (i.e., redox stimuli oxidise select targets); and (3) heterogenous (i.e., target responses vary markedly). RedoxiFluor is a powerful new tool for advancing a far-reaching and influential field: protein thiol redox biology.

Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein–protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As3+ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As3+ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As3+ in various biological and environmental systems.

Green self-immolative polymer: molecular antenna to collect and propagate the signal for zymogen activation

Chemical zymogens of three different types were established herein around protein cysteinome, in each case converting the protein thiol into a disulfide linkage: zero length Z0, polyethylene glycol based ZPEG, and ZLA that features a fast-depolymerizing fuse polymer. The latter was a polydisulfide based on a naturally occurring water-soluble lipoic acid. Three zymogen designs were applied to cysteinyl proteases and a kinase and in each case, enzymatic activity was successfully masked in full and reactivated by small molecule reducing agents. However, only ZLA could be reactivated by protein activators, demonstrating that the macromolecular fuse escapes the steric bulk created by the protein globule, collects activation signal in solution, and relays it to the enzyme active site. This afforded first-in-class chemical zymogens that are activated via protein-protein interactions. For ZLA, we also document a "chain transfer" bioconjugation mechanism and a unique zymogen exchange reaction between two proteins.