Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 µM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) as a user-friendly system to detect SARS-CoV-2 infection: a multicentric study.

Although reverse transcriptase quantitative PCR remains the gold standard to perform viral detection, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is already used to perform diagnosis of various infections. This work reports the results of a multicentric study performed in Sicily to evaluate the diagnostic power of an RT-LAMP kit for the diagnosis of SARS-CoV-2 infection on a total of 551 samples collected in January and February 2021, revealing sensitivity, specificity, accuracy, positive and negative predictive values ≥95%. Our results suggest the potential employment of this kit as a screening test to be used where fast and reliable results are demanded without the need for expensive instruments and highly-skilled personnel.

A new erythrocyte-based biochemical approach to predict the antiproliferative effects of heterocyclic scaffolds: The case of indolone.

BACKGROUND: The indole core is a key structural feature of many natural products and biomolecules with broad spectrum chemotherapeutic properties. Some of us have recently synthesized a library of biologically promising indolone-based compounds. The present study focuses on the effects of one of them, namely DPIT, on human erythrocytes. METHODS: We have examined the influence of DPIT on band 3 protein, intracellular ATP concentration and transport, caspase 3 activation, metabolic adaptation and membrane stability. RESULTS: Our study elucidates that DPIT, intercalated into the phospholipid bilayer, decreases the anion transport, the intracellular ATP concentration and the cytosolic pH, inducing a direct activation of caspase 3. CONCLUSIONS: Starting from the metabolic similarity between erythrocytes and cancer cells, we investigate how the metabolic derangements and membrane alterations induced by selected heterocycles could be related to the antiproliferative effects. GENERAL SIGNIFICANCE: Our work aims to propose a new model of study to predict the antiproliferative effects of heterocyclic scaffolds, pointing out that only one of the listed conditions would be unfavorable to the life cycle of neoplastic cells.

The Role of Hypoxia and Hypoxia Signaling in Skeletal Muscle Physiology.

Hypoxia and hypoxia signaling play an integral role in regulating skeletal muscle physiology. Environmental hypoxia and tissue hypoxia in muscles cue for their appropriate physiological response and adaptation, and cause an array of cellular and metabolic changes. In addition, muscle stem cells (satellite cells), exist in a hypoxic state, and this intrinsic hypoxic state correlates with their quiescence and stemness. The mechanisms of hypoxia-mediated regulation of satellite cells and myogenesis are yet to be characterized, and their seemingly contradicting effects reported leave their exact roles somewhat perplexing. This review summarizes the recent findings on the effect of hypoxia and hypoxia signaling on the key aspects of muscle physiology, namely, stem cell maintenance and myogenesis with a particular attention given to distinguish the intrinsic versus local hypoxia in an attempt to better understand their respective regulatory roles and how their relationship affects the overall response. This review further describes their mechanistic links and their possible implications on the relevant pathologies and therapeutics.

In Vitro Simulated Hemoperfusion on Seraph®-100 as a Promising Strategy to Counteract Sepsis.

Blood purification represents a treatment option for sepsis, improving inflammation and the hyper-activated immune system. This study investigates the binding efficacy of Seraph®-100 against 108 CFU/mL of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) during a simulated hemoperfusion treatment. The fluorescence-activated cell sorting (FACS) technique was used to evaluate the bacteria reduction, whereas kinetic analysis and cultures revealed bacterial detection and counting at established time points. At the end of the experiment, the filter was cut at three different levels, obtaining suspensions for cultures and scanning electron microscopy (SEM) analyses. The FACS technique revealed a 78.77% reduction of the total bacterial load at the end of the treatment, with maximum filter sequestration occurring in the first 30 min of the treatment. Non-linear regression analysis of kinetic experiments (T0-240 min) highlighted a lower growth rate of S. aureus than the other two Gram bacteria, demonstrating a greater affinity without influencing a reduction rate of 99% for all three bacteria. The analyses of the suspension aliquots of the filter sections confirmed these data, revealing 1 × 108 CFU/mL, equal to the initial bacterial charge. Furthermore, the filter head adsorbed approximately 50% of bacteria, whereas the remaining amount was equally distributed between the body and the tail, as corroborated by SEM analysis. In conclusion, Seraph®-100 adsorbed 108 CFU/mL of S. aureus, E. coli, and P. aeruginosa during an in vitro simulated hemoperfusion session.

Free Light Chains, High Mobility Group Box 1, and Mortality in Hemodialysis Patients.

Background: Uremic toxins are associated with immune dysfunction and inflammation. The inadequate removal by hemodialysis (HD) of serum free light chains (FLCs) determines their accumulation. This study evaluated FLCs in HD patients, analyzing their relations with other biomarkers, such as serum high mobility group box 1 (HMGB1). Methods: FLC and HMGB1 were evaluated in a cohort of 119 HD patients. κFLC and λFLC were summated to give a combined (c) FLC concentration. Patients were followed prospectively until the end of the observation period of four years, or until the endpoint: the patient's death. Results: cFLC values in HD patients were 244.4 (197.9−273.5) mg/L. We detected a significant reduction in CD8+ cells and a decreased CD4+/CD8+ ratio. HMGB1 levels were 94.5 (55−302) pg/mL. After multivariate analysis, cFLCs correlated with ß2-microglobulin and the CD4+/CD8+ ratio. Subjects with cFLC values above 263 mg/L and with sHMGB1 values < 80 pg/mL experienced a significantly faster evolution to the endpoint (mean follow-up time to progression of 27.5 and 28.5 months, respectively; p < 0.001). After an adjusted multivariate Cox analysis, cFLCs were associated with 11% increased risk of death, whereas low sHMGB1 increased this risk by 5%. Conclusions: cFLCs and HMGB1 reflect the inflammation and immune dysfunction in HD patients representing two strong and independent risk markers of mortality.

Short-Term Effects of Chlorpromazine on Oxidative Stress in Erythrocyte Functionality: Activation of Metabolism and Membrane Perturbation.

The purpose of this paper is to focus on the short-term effects of chlorpromazine on erythrocytes because it is reported that the drug, unstable in plasma but more stable in erythrocytes, interacts with erythrocyte membranes, membrane lipids, and hemoglobin. There is a rich literature about the side and therapeutic effects or complications due to chlorpromazine, but most of these studies explore the influence of long-term treatment. We think that evaluating the short-term effects of the drug may help to clarify the sequence of chlorpromazine molecular targets from which some long-term effects derive. Our results indicate that although the drug is primarily intercalated in the innermost side of the membrane, it does not influence band 3 anionic flux, lipid peroxidation, and protein carbonylation processes. On the other hand, it destabilizes and increases the autooxidation of haemoglobin, induces activation of caspase 3, and, markedly, influences the ATP and reduced glutathione levels, with subsequent exposure of phosphatidylserine at the erythrocyte surface. Overall our observations on the early stage of chlorpromazine influence on erythrocytes may contribute to better understanding of new and interesting characteristics of this compound improving knowledge of erythrocyte metabolism.

Alterations in Red Blood Cell Functionality Induced by an Indole Scaffold Containing a Y-Iminodiketo Moiety: Potential Antiproliferative Conditions.

We have recently proposed a new erythrocyte-based model of study to predict the antiproliferative effects of selected heterocyclic scaffolds. Starting from the metabolic similarity between erythrocytes and cancer cells, we have demonstrated how the metabolic derangement induced by an indolone-based compound (DPIT) could be related to its antiproliferative effects. In order to prove the validity of our biochemical approach, in the present study the effects on erythrocyte functionality of its chemical precursor (PID), whose synthesis we reported, were investigated. The influence of the tested compound on band 3 protein (B3), oxidative state, ATP efflux, caspase 3, metabolism, intracellular pH, and Ca(2+) homeostasis has been evaluated. PID crosses the membrane localizing into the cytosol, increases anion exchange, induces direct caspase activation, shifts the erythrocytes towards an oxidative state, and releases less ATP than in normal conditions. Analysis of phosphatidylserine externalization shows that PID slightly induces apoptosis. Our findings indicate that, due to its unique features, erythrocyte responses to exogenous molecular stimuli can be fruitfully correlated at structurally more complex cells, such as cancer cells. Overall, our work indicates that erythrocyte is a powerful study tool to elucidate the biochemical/biological effects of selected heterocycles opening considerable perspectives in the field of drug discovery.

Involvement of acetylcholinesterase and protein kinase C in the protective effect of caffeine against ß-amyloid-induced alterations in red blood cells.

It is well known the role of oxidative stress in the pathophysiology of Alzheimer's disease (AD) and of other neurodegenerative pathologies. We have previously documented that Amyloid beta peptide (1-42) (Abeta) dependent-oxidative modifications affect red blood cell (RBC) morphology and function. Experimental studies show that caffeine (CF) consumption is inversely correlated with AD. In this study, we investigated the role played by RBC in the protective mechanism elicited by CF against Abeta mediated toxicity. PS exposure levels by FACS analysis, as well as protein band 3 functionality analysis, indicated that CF at 100 µM protected against Abeta-mediated membrane alterations, which are known to occur in AD. Moreover, CF counteracts inhibition of ATP release from RBC by Abeta, restoring its ability to modulate vasodilation. Concurrently, analysis of protein kinase C (PKC) and caspase 3 activities, responsible for cytoskeleton alterations, revealed that unlike to caspase 3, PKCα activation induced by Abeta was fully abolished by CF through a mechanism involving Acetylcholinesterase (AChE), located on external face of RBC plasma membrane. These results provide support for the hypothesis concerning the protective role of CF in AD patients could include also a peripheral mechanism involving RBC.

Identification of paraoxonase 3 gene (PON3) missense mutations in a population of southern Italy.

PON gene family includes at least three members termed PON1, PON2 and PON3, and it is mapped on human chromosome 7q21-q22. PON1 and PON3 gene products are constituents of high density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. PON1 and PON2 genes have missense polymorphisms, but, to date, no missense variants are reported in PON3 gene. In this work we explored the existence of genetic variants within the PON3 coding sequences. Five point mutations were identified by direct sequencing of genomic DNA derived from 250 randomly selected DNA samples of 1143 blood donors living in southern Italy. Three were silent mutations, while two were missense mutations that give rise to amino acid substitutions at positions 311 (S>T) and 324 (G>D). The missense variations in the DNA of the 1143 samples had frequencies of 0.22% (5 out of 2286 alleles) for the S311T mutation, and 0.57% (13 out of 2286 alleles) for the G324D mutation. The effect of these variants on the metabolic activity of paraoxonase 3 remains to be further evaluated.