Precipitating factors of bradycardia after remdesivir administration: ICU admission and cutoff value for declining heart rate.

BACKGROUND: Despite increasing concerns about the association between remdesivir and bradycardia in severe coronavirus disease 2019 (COVID-19) patients receiving remdesivir, information on its clinical course and precipitating factors is limited. Our aim was to investigate possible triggers of bradycardia after remdesivir administration. METHODS: We retrieved the medical records of hospitalized severe and critical COVID-19 patients who received remdesivir from May 1, 2021 to June 30, 2021. Bradycardia was defined as two episodes of a heart rate (HR) < 60 bpm in 24 h. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the discriminability of heart rate pattern on the occurrence of bradycardia. The precipitating factors of bradycardia were examined by a logistic regression model. RESULTS: Regardless of bradycardia status, the median heart rate dropped during remdesivir treatment (from 85 to 72 bpm, p < 0.001), with the heart rate dropping considerably within the first two days of remdesivir treatment. Among various heart rate descriptors, HR ratiomin (d2-d1) had the best discrimination (AUC = 0.7336), and a reduction in HR ratiomin (d2-d1) by 14.65% was associated with bradycardia. Intensive care unit (ICU) admission was associated with an increased risk of bradycardia (odds ratio: 3.41; 95% CI: 1.12-10.41). CONCLUSIONS: In severe COVID-19 patients receiving remdesivir, the risks of bradycardia were influenced by a substantial reduction in heart rate during the first two days of remdesivir treatment and ICU admission. These findings suggest that clinical practitioners should intensively monitor heart rates during remdesivir treatment.

ZnO Nanoparticles Induced Caspase-Dependent Apoptosis in Gingival Squamous Cell Carcinoma through Mitochondrial Dysfunction and p70S6K Signaling Pathway.

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, food additives, pigments, rubber manufacture, and electronic materials. Several studies have shown that ZnO-NPs inhibit cell growth and induce apoptosis by the production of oxidative stress in a variety of human cancer cells. However, the anti-cancer property and molecular mechanism of ZnO-NPs in human gingival squamous cell carcinoma (GSCC) are not fully understood. In this study, we found that ZnO-NPs induced growth inhibition of GSCC (Ca9-22 and OECM-1 cells), but no damage in human normal keratinocytes (HaCaT cells) and gingival fibroblasts (HGF-1 cells). ZnO-NPs caused apoptotic cell death of GSCC in a concentration-dependent manner by the quantitative assessment of oligonucleosomal DNA fragmentation. Flow cytometric analysis of cell cycle progression revealed that sub-G1 phase accumulation was dramatically induced by ZnO-NPs. In addition, ZnO-NPs increased the intracellular reactive oxygen species and specifically superoxide levels, and also decreased the mitochondrial membrane potential. ZnO-NPs further activated apoptotic cell death via the caspase cascades. Importantly, anti-oxidant and caspase inhibitor clearly prevented ZnO-NP-induced cell death, indicating the fact that superoxide-induced mitochondrial dysfunction is associated with the ZnO-NP-mediated caspase-dependent apoptosis in human GSCC. Moreover, ZnO-NPs significantly inhibited the phosphorylation of ribosomal protein S6 kinase (p70S6K kinase). In a corollary in vivo study, our results demonstrated that ZnO-NPs possessed an anti-cancer effect in a zebrafish xenograft model. Collectively, these results suggest that ZnO-NPs induce apoptosis through the mitochondrial oxidative damage and p70S6K signaling pathway in human GSCC. The present study may provide an experimental basis for ZnO-NPs to be considered as a promising novel anti­tumor agent for the treatment of gingival cancer.

Examining the inhibitory potency of food additive fast green FCF against amyloid fibrillogenesis under acidic conditions.

More than thirty human proteins and/or peptides can fold incorrectly to form amyloid deposits associated with several protein aggregation diseases. No cure is currently available for treating these diseases. This work is aimed at examining the inhibitory potency of fast green FCF, a biocompatible dye, toward the fibrillogenesis/aggregation of lysozyme. As verified by ThT binding assay along with transmission electron microscopy, fast green FCF was observed to suppress the generation of lysozyme fibrils in a concentration-dependent manner. We next used circular dichroism absorption spectroscopy, ANS fluorescence spectroscopy, and SDS-PAGE to characterize the structural alterations in lysozyme samples upon the addition of fast green FCF. Furthermore, experiments with the addition of fast green FCF at different time points of incubation showed that fast green FCF also exhibited disaggregating activity against the preformed/existing lysozyme fibrils. We believe that the results from this study suggest a potential therapeutic role of biocompatible molecules in treating or preventing protein aggregation diseases.

Experimentally validated novel inhibitors of Helicobacter pylori phosphopantetheine adenylyltransferase discovered by virtual high-throughput screening.

Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.

The impact of V30A mutation on transthyretin protein structural stability and cytotoxicity against neuroblastoma cells.

Single point mutations in the transthyretin (TTR) gene may cause a hereditary neurodegenerative disease termed familial amyloidotic polyneuropathy (FAP) due to accelerated deposition of amyloid fibrils, resulting in peripheral and autonomic nervous system dysfunction. Recently, we found a Chinese FAP family involving a TTR V30A mutation. To understand the pathogenic mechanisms of this V30A TTR, we investigated the effects of this mutation on TTR quaternary and tertiary structural stabilities and cytotoxicities against neuroblastoma cells along with the most common variant V30M TTR and the wild-type (WT) TTR. Our results showed that the V30A mutation impaired the thermodynamic and kinetic stabilities of the TTR protein by increasing the extent and rate of tetramer dissociation and unfolded monomer and amyloid fibril formation, even to a greater extent than the V30M mutation under several experimental conditions. Further, an obviously cytotoxic effect of the V30A TTR on the human neuroblastoma cell line, IMR-32, was observed. The V30A TTR induced apoptosis and autophagy concomitant with the accumulation of reactive oxygen species (ROS) and DNA double-strand breaks, reflected in the induction of phosphor-H2A.X. These results suggest that the V30A mutation in the TTR gene promotes the formation of unfolded monomers and amyloid fibrils, which potentially contribute to the increased neurotoxicity and the pathology associated with this FAP family.

Using isothermal titration calorimetry to real-time monitor the heat of metabolism: a case study using PC12 cells and Aß(1-40).

Isothermal titration calorimetry (ITC) is one of the most powerful means for direct determination of thermodynamic information associated with most physiochemical and biological processes. The deposition and aggregation of ß-amyloid (Aß) on cell membranes was considered as one of the primary factors in having Alzheimer's disease (AD). Recently, a growing body of evidence has suggested that plasma membrane could accelerate the accumulation of Aß on the plasma membranes. However, the mechanism of AD is still a controversial issue. This study provided a biothermodynamic approach to real-time monitor the heat of metabolism involved in the co-incubation of PC12 cells and Aß(1-40) by ITC. The effects of Aß conformation and concentration of oligomeric Aß on cytotoxicity were successfully distinguished by ITC. This approach with rapid and direct measurement may provide not only real-time information for the effects of Aß species on living cells but also a platform for the screening of drug candidates for AD.

Investigating the effects of sodium dodecyl sulfate on the aggregative behavior of hen egg-white lysozyme at acidic pH.

The research presented here is aimed at examining the effects of sodium dodecyl sulfate on the aggregative behavior of hen egg-white lysozyme at pH 2.0. Through various spectroscopic techniques, dynamic light scattering, and electron microscopy, we first demonstrated that SDS exhibited a biphasic effect on lysozyme fibrillation. The presence of SDS at higher concentrations (e.g., 0.25, 5.00, or 20.00 mM SDS) was found to suppress fibril formation of lysozyme whereas fibrillogenic lysozyme-SDS ensemble containing beta-sheet-rich conformation was observed upon the addition of lower concentrations of SDS (e.g., 0.00, 0.06, or 0.1mM SDS). Next, our equilibrium urea-unfolding data revealed that lysozyme samples with higher SDS concentrations showed superior thermodynamic stabilities over the ones with no or lower levels of SDS. Finally, the correlation between SDS concentration and lysozyme aggregative/fibrillogenic propensity and the underlying interacting mechanism were further explored using surface tensiometry and isothermal titration calorimetry. We believe the outcome from this work may not only help decipher the molecular mechanism of amyloid fibrillation, but also shed light on a rational design of potential therapeutic strategies for amyloid pathology.

Recent trends and some applications of isothermal titration calorimetry in biotechnology.

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.

Dynamic fluorescence imaging analysis to investigate the cholesterol recruitment in lipid monolayer during the interaction between beta-amyloid (1-40) and lipid monolayers.

Extracellular beta-amyloid (Abeta) deposit is considered as one of the primary factors in inducing Alzheimer's disease (AD). However, the mechanism of Abeta deposition on the cell membrane and the induced cytotoxicity are still unclear. On the basis of the previous reports and results, we propose the "Recruiting Hypothesis" on the interaction between the plasma membrane and Abeta. Recently, many studies focused on cholesterol, which is considered as an important factor for AD. The most challenging issue in studying the cholesterol is non-ideal mixing behavior and non-dynamic analysis. In the present study, we investigated the cholesterol recruitment in the lipid monolayer during the interaction between beta-amyloid peptides Abeta (1-40) and lipid monolayers by dynamic fluorescent imaging analysis. Results from lipid monolayer trough studies showed that the rate of Abeta adsorption onto lipid monolayer is mainly due to the electrostatic effect which is sensitive to the lipid monolayer composition. From the fluorescence imaging analysis, the interaction of Abeta with lipid monolayer containing negative charge lipid and cholesterol brings out the recruiting behavior of the cholesterol and reduces the fluidity of lipid. The present study not only demonstrates the technical application for monitoring the dynamic molecular behaviors at the interface but also reveals the roles to distinguish lipid molecules on the Abeta-membrane interaction.

Examining the levels of ganglioside and cholesterol in cell membrane on attenuation the cytotoxicity of beta-amyloid peptide.

The deposition of beta-amyloid (Abeta) on cell membranes is considered as one of the primary factors in having Alzheimer's disease (AD). Recent studies have suggested that certain components of plasma membrane, ganglioside and cholesterol could accelerate the accumulation of Abeta on the plasma membranes. However, the effect of cholesterol and ganglioside (GM1) on Abeta cytotoxicity is still a controversial issue. The aim of this study is to understand the roles of GM1 and cholesterol in AD by using PC12, a neuron-like cell. The effects of the sequence, conformation, and concentration of Abeta on cytotoxicity were also investigated. Monomeric Abeta could attack the plasma membrane resulting in cytotoxicity, however, fibrillar Abeta was found to be less toxic. Our results showed that Abeta (1-40) was more toxic than Abeta (25-35) and the cytotoxicity of Abeta was proportional to its concentration. Besides, the depletion of GM1 from plasma membrane, it would block the Abeta-induced cytotoxicity. Decreasing the cholesterol level by around 30% could attenuate the cytotoxicity of Abeta. These findings validate our idea that the cholesterol could stabilize the lateral pressure derived from the formation of GM1-Abeta complex on the membrane surface. Furthermore, both GM1 and cholesterol are essential in mechanism of Abeta accumulation and could modulate the cytotoxicity of monomeric Abeta.

Investigation of the mechanism of beta-amyloid fibril formation by kinetic and thermodynamic analyses.

Extracellular beta-amyloid (A beta) deposit is considered as one of the primary factors that induce Alzheimer's disease (AD). The effects of various environmental factors, including temperature, ionic strength, and pH, on A beta (1-40) aggregation mechanisms were investigated in this study by spectrometry, isothermal titration calorimetry (ITC), and hydrophobic fluorescence assay. In the aggregation process, the secondary structure of A beta (1-40) transforms to the beta-sheet conformation, which could be described as a two-state model. As the temperature and ionic strength increase, the conformation of A beta converts to the beta-sheet structure with an increased rate. Results of circular dichroism monitoring demonstrate that the rate constant of nucleation is smaller than that of elongation, and the nucleation is the rate-determining step during the overall A beta aggregation. The beta-sheet structure was stabilized by hydrophobic forces, as revealed by the ITC measurements. The different structural aggregates and forming pathways could be identified and discriminated at high and low ionic strengths, resulting in distinctive fibril conformations. Furthermore, the thermodynamic analysis shows that hydrophobic interaction is the major driving force in the nucleation step. Our study provides an insight into the discriminative mechanisms of beta-amyloid aggregation via kinetics and thermodynamics, especially the first reported thermodynamics information obtained by ITC.

Microcalorimetrics studies of the thermodynamics and binding mechanism between L-tyrosinamide and aptamer.

In recent years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complex interactions. In some cases, however, the aptamer complex binding behavior and mechanism are not clearly understood, especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC) and circular dichroism (CD) were useful tools for studying the fundamental binding mechanism between a DNA aptamer and L-tyrosinamide (L-TyrNH2). To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value, analogue of L-TyrNH2, and metal ion were carried out. The thermodynamic signature along with the coupled CD spectral change suggest that this binding behavior is an enthalpy-driven process, and the aptamer has a conformational change from B-form to A-form. The results showed that the interaction is an induced fit binding, and the driving forces in this binding behavior may include electrostatic interactions, hydrophobic effects, hydrogen bonding, and the binding-linked protonation process. The amide group and phenolic hydroxyl group of the L-TyrNH2 play a vital role in this binding mechanism. In addition, it should be noted that Mg(2+) not only improves binding affinity but also helps change the structure of the DNA aptamer.

Kinetics and enthalpy measurements of interaction between beta-amyloid and liposomes by surface plasmon resonance and isothermal titration microcalorimetry.

The objective of this research is to understand the interaction mechanism of beta-amyloid (Abeta) with cell and were basically divided into two parts. The first part focused on the time-dependent structural changes of Abeta (1-40) by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence assay, and atomic force microscopy (AFM). The second part emphasized the kinetics and enthalpy of interaction between Abeta (1-40) and liposome by surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). Results obtained from CD, ThT and AFM confirmed the formation of 1 microm fibril after single day incubation. The driving force of kinetic interaction between Abeta and liposomes was revealed by SPR to be electrostatics. Further studies indicated that fresh Abeta has high GM1 affinity. Besides, addition of cholesterol to the liposome could alter membrane fluidity and affect the interactions of fresh Abeta with liposomes especially in the amount of Abeta absorbed and preserving the structure of liposome after adsorbing. Hydrophobicity was found to be the driving force leading to the interaction between Abeta fibrils and liposomes. These reactions are endothermic as supported by ITC measurements. When the composition of liposomes is zwitterionic lipids, the interaction of Abeta with liposomes is predominantly hydrophobic force. In contrast, the driving force of interaction of charged lipids with Abeta is electrostatic.

Characterization of the interaction of galectin-1 with sodium arsenite.

We previously showed that galectin-1 (GAL1) is an arsenic-binding protein. In the current study, we further characterize the interaction of GAL1 with sodium arsenite (As(III)). The GALl-As(III) complex was prepared from the cell extracts of GAL1-transfected Escherichia coli (E. coli) that were pretreated with As(III). The results of the circular dichroism (CD) spectrum of GAL1-As(III) exhibited a negative signal at around 205-210 nm, whereas that of GAL1 showed a negative signal at around 215-220 nm. This shift in the CD spectrum is indicative of a substantial change in the secondary structure arising from the binding of As(III) to the GAL1 protein. The UV absorptive spectrum of the GAL1-As(III) complex was significantly lower than that of GAL1 itself. A mobility shift binding assay showed that the GAL1-As(III) complex migrated closer than GAL1 toward the anode. Capillary electrophoretic analysis also showed that As(III) binding decreased the mobility of GAL1. These results further confirmed the structural change of the GAL1 complex with As(III). Furthermore, isothermal titration microcalometric studies showed that As(III) titration into the GAL1 protein solution was an endothermic process with absorption enthalpy (DeltaH(abs)) around 8-10 kJ/mol As(III). The affinity constant (K(d)) of As(III) toward GAL1 was around 8.239 +/- 2.627 microM as estimated by tryptophan (Trp) fluorescence quenching. However, the binding of As(III) did not significantly affect the biological activity of GAL1, since the GAL1-As(III) complex only partially lost its lectin activity. In addition, we show that GAL1-transfected KB cells accumulated more arsenic than did the parental cells. Taken together, these results suggest that GAL1 might serve as a target protein of As(III) in vivo, and the binding of GAL1 with As(III) could interfere with the excretion of As(III).