Chalcones as Scavengers of HOCl and Inhibitors of Oxidative Burst: Structure-Activity Relationship Studies.

AIMS: This study evaluates the ability of chalcones to scavenge hypochlorous acid (HOCl) and modulate oxidative burst. BACKGROUND: The chemistry of chalcones has long been a matter of interest to the scientific community due to the phenolic groups often present and to the various replaceable hydrogens that allow the formation of a broad number of derivatives. Due to this chemical diversity, several biological activities have been attributed to chalcones, namely anti-diabetic, anti-inflammatory and antioxidant. OBJECTIVES: Evaluate the ability of a panel of 34 structurally related chalcones to scavenge HOCl and/or suppress its production through the inhibition of human neutrophils' oxidative burst, followed by the establishment of the respective structure-activity relationships. METHODS: The ability of chalcones to scavenge HOCl was evaluated by fluorimetric detection of the inhibition of dihydrorhodamine 123 oxidation. The ability of chalcones to inhibit neutrophils' oxidative burst was evaluated by chemiluminometric detection of the inhibition of luminol oxidation. RESULTS: It was observed that the ability to scavenge HOCl depends on the position and number of hydroxy groups on both aromatic rings. Chalcone 5b was the most active with an IC50 value of 1.0 ± 0.1 µM. The ability to inhibit neutrophils' oxidative burst depends on the presence of a 2'-hydroxy group on A-ring and on other substituents groups, e.g. methoxy, hydroxy, nitro and/or chlorine atom( s) at C-2, C-3 and/or C-4 on B-ring, as in chalcones 2d, 2f, 2j, 2i, 4b, 2n and 1d, which were the most actives with IC50 values ranging from 0.61 ± 0.02 µM to 1.7 ± 0.2 µM. CONCLUSION: The studied chalcones showed high activity at a low micromolar range, indicating their potential as antioxidant agents and to be used as a molecular structural scaffold for the design of new anti-inflammatory compounds.

Influence of hypoxia on biochemical aspects and on expression of genes related to oxygen-homeostasis of the Amazonian cichlid Astronotus ocellatus (Agassiz, 1831).

Variations in dissolved oxygen levels are common in the Amazonian aquatic environments and the aquatic organisms that inhabit these environments developed a variety of adaptive responses to deal with such conditions. Some Amazonian fish species are tolerant to low oxygen levels and the cichlid Astronotus ocellatus is one of the most hypoxia-tolerant species. Herein, we aimed to unveil the biochemical and molecular responses that A. ocellatus presents when submitted to hypoxia. Hypoxia indicators were measured, such as plasma glucose, plasma lactate, hepatic glycogen and relative transcript levels of prolyl hydroxylase 2 (phd2) and hypoxia-inducible factor-1α (hif-1α) in juveniles of approximately 50 g exposed to 1, 3, and 5 hours of hypoxia (0.7 mg O2.L-1), followed by 3 hours of recovery in normoxia (6 mg O2.L-1). Fish exposed to hypoxia reduced liver glycogen levels within 3 hours of hypoxia, when comparing with 1 hour, and increased plasma glucose and lactate. Under the same condition, phd2 transcripts levels increased in gills, but decreased in liver. In contrast, hypoxia did not affect relative gene expression of hif-1α in both tissues. Based on the transcription pattern of phd2, these results showed that liver and gills of A. ocellatus have different molecular strategies to cope with environmental hypoxia.

Convalescent plasma-mediated resolution of COVID-19 in a patient with humoral immunodeficiency.

Convalescent plasma (CP) is widely used to treat COVID-19, but without formal evidence of efficacy. Here, we report the beneficial effects of CP in a severely ill COVID-19 patient with prolonged pneumonia and advanced chronic lymphocytic leukemia (CLL), who was unable to generate an antiviral antibody response of her own. On day 33 after becoming symptomatic, the patient received CP containing high-titer (ID50 > 5,000) neutralizing antibodies (NAbs), defervesced, and improved clinically within 48 h and was discharged on day 37. Hence, when present in sufficient quantities, NAbs to SARS-CoV-2 have clinical benefit even if administered relatively late in the disease course. However, analysis of additional CP units revealed widely varying NAb titers, with many recipients exhibiting endogenous NAb responses far exceeding those of the administered units. To obtain the full therapeutic benefits of CP immunotherapy, it will thus be important to determine the neutralizing activity in both CP units and transfusion candidates.

Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection.

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.

Optimization of Experimental Settings for the Assessment of Reactive Oxygen Species Production by Human Blood.

The purpose of an experimental design is to improve the productivity of experimentation. It is an efficient procedure for planning experiments, so the data obtained can be analyzed to yield a valid and objective conclusion. This approach has been used as an important tool in the optimization of different analytical approaches. A D-optimal experimental design was used here, for the first time, to optimize the experimental conditions for the detection of reactive oxygen species (ROS) produced by human blood from healthy donors, a biological matrix that better resembles the physiologic environment, following stimulation by a potent inflammatory mediator, phorbol-12-myristate-13-acetate (PMA). For that purpose, different fluorescent probes were used, as 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and 10-acetyl-3,7-dihydroxyphenoxazine (amplex red). The variables tested were the human blood dilution, and the fluorescent probe and PMA concentrations. The experiments were evaluated using the Response Surface Methodology and the method was validated using specific compounds. This model allowed the search for optimal conditions for a set of responses simultaneously, enabling, from a small number of experiments, the evaluation of the interaction between the variables under study. Moreover, a cellular model was implemented and optimized to detect the production of ROS using a yet nonexplored matrix, which is human blood.

Calcium Pathways in Human Neutrophils-The Extended Effects of Thapsigargin and ML-9.

In neutrophils, intracellular Ca2+ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca2+-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca2+ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca2+ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca2+ pathways, that, in the future, should be further explored.

Immunomodulatory Effects of Flavonoids in the Prophylaxis and Treatment of Inflammatory Bowel Diseases: A Comprehensive Review.

Inflammatory Bowel Diseases (IBD) comprised of two disorders of idiopathic chronic intestinal inflammation that affect about three million people worldwide: Crohn's disease and ulcerative colitis. Nowadays, the first-line of treatment for patients with mild to moderate symptoms of IBD is comprised of corticosteroids, immunosuppressants, antibiotics, and biological agents. Unfortunately, none of these drugs are curative, and their long-term use may cause severe side effects and complications. Almost 40% of IBD patients use alternative therapies to complement the conventional one, and flavonoids are gaining attention for this purpose. The biological properties of flavonoids are well documented and their antioxidant and anti-inflammatory activities have been arousing attention in the scientific community. Flavonoids are the most widely distributed polyphenols in plants and fruits, making part of the human diet. Taking into account that all ingested flavonoids are expected to exert biological actions at the gastrointestinal level, research on the modulatory effect of these compounds in IBD is of paramount importance. This review intends to summarize, in an integrated and comprehensive form, the effect of flavonoids, both in vitro and in vivo, in the different phases of the characteristic IBD inflammatory network.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (µ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL-1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 µg mL-1 (corresponding to 5.0-60 mg mL-1 in undiluted samples), with a detection limit of 33 µg mL-1 (1.7 mg mL-1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

A biophysical approach to daunorubicin interaction with model membranes: relevance for the drug's biological activity.

Daunorubicin is extensively used in chemotherapy for diverse types of cancer. Over the years, evidence has suggested that the mechanisms by which daunorubicin causes cytotoxic effects are also associated with interactions at the membrane level. The aim of the present work was to study the interplay between daunorubicin and mimetic membrane models composed of different ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM) and cholesterol (Chol). Several biophysical parameters were assessed using liposomes as mimetic model membranes. Thereby, the ability of daunorubicin to partition into lipid bilayers, its apparent location within the membrane and its effect on membrane fluidity were investigated. The results showed that daunorubicin has higher affinity for lipid bilayers composed of DMPC, followed by DMPC : SM, DMPC : Chol and lastly by DMPC : SM : Chol. The addition of SM or Chol into DMPC membranes not only increases the complexity of the model membrane but also decreases its fluidity, which, in turn, reduces the amount of anticancer drug that can partition into these mimetic models. Fluorescence quenching studies suggest a broad distribution of the drug across the bilayer thickness, with a preferential location in the phospholipid tails. The gathered data support that daunorubicin permeates all types of membranes to different degrees, interacts with phospholipids through electrostatic and hydrophobic bonds and causes alterations in the biophysical properties of the bilayers, namely in membrane fluidity. In fact, a decrease in membrane fluidity can be observed in the acyl region of the phospholipids. Ultimately, such outcomes can be correlated with daunorubicin's biological action, where membrane structure and lipid composition have an important role. In fact, the results indicate that the intercalation of daunorubicin between the phospholipids can also take place in rigid domains, such as rafts that are known to be involved in different receptor processes, which are important for cellular function.

Influence of doxorubicin on model cell membrane properties: insights from in vitro and in silico studies.

Despite doxorubicin being commonly used in chemotherapy there still remain significant holes in our knowledge regarding its delivery efficacy and an observed resistance mechanism that is postulated to involve the cell membrane. One possible mechanism is the efflux by protein P-gp, which is found predominantly in cholesterol enriched domains. Thereby, a hypothesis for the vulnerability of doxorubicin to efflux through P-gp is its enhanced affinity for the ordered cholesterol rich regions of the plasma membrane. Thus, we have studied doxorubicin's interaction with model membranes in a cholesterol rich, ordered environment and in liquid-disordered cholesterol poor environment. We have combined three separate experimental protocols: UV-Vis spectrophotometry, fluorescence quenching and steady-state anisotropy and computational molecular dynamics modeling. Our results show that the presence of cholesterol induces a change in membrane structure and doesn't impair doxorubicin's membrane partitioning, but reduces drug's influence on membrane fluidity without directly interacting with it. It is thus possible that the resistance mechanism that lowers the efficacy of doxorubicin, results from an increased density in membrane regions where the efflux proteins are present. This work represents a successful approach, combining experimental and computational studies of membrane based systems to unveil the behavior of drugs and candidate drug molecules.

Chlorinated Flavonoids Modulate the Inflammatory Process in Human Blood.

Flavonoids are known to react with neutrophil-generated hypochlorous acid (HOCl) at inflammation loci to form stable mono- and dichlorinated products. Some of these products have been shown to retain or even enhance their inflammatory potential, but further information is required in a broader approach to inflammatory mechanisms. In that sense, we performed an integrated evaluation on the anti-inflammatory potential of a panel of novel chlorinated flavonoids and their parent compounds, in several steps of the complex inflammatory cascade, namely, in the activity of cyclooxygenase (COX)-1 and COX-2, and in the production of cytokines [interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)], and the chemokine, IL-8, as well as in the production of reactive species, using human whole blood as a representative in vitro model, establishing, whenever possible, a structure-activity relationship. Although luteolin was the most active compound, chlorinated flavonoids demonstrated a remarkable pattern of activity for the resolution of the inflammatory processes. Our results demonstrated that 6-chloro-3',4',5,7-tetrahydroxyflavone deserves scientific attention due to its ability to modulate the reactive species and cytokines/chemokine production. In this regard, the therapeutic potential of flavonoids' metabolites, and in this particular case the chlorinated flavonoids, should not be neglected.

The daunorubicin interplay with mimetic model membranes of cancer cells: A biophysical interpretation.

The present work aimed to study the interactions between the anticancer drug daunorubicin and lipid membrane mimetic models of cancer cells composed by their most representative classes of phospholipids, with different degrees of complexity. Regarding these anticancer drug-membrane interactions, several biophysical parameters were assessed using liposomes (LUVs) composed of different molar ratios of DMPC, DOPC, DPPS, DOPE and Chol. In this context, daunorubicin's membrane concentration was determined by calculating its partition coefficient (Kp) between liposomes and water using derivative UV/vis spectrophotometry at 37°C and pH6.3, a typical tumoral microenvironment. Characterization of the zeta potential of such model membranes, in both the absence and presence of the compound, was accomplished through Electrophoretic Light Scattering (ELS). Fluorescence quenching studies, which determine the location of the drug within the bilayer, were carried out using liposomes labelled with DPH and TMA-DPH, fluorescent probes with known membrane position. Temperature dependent steady-state anisotropy assays were also performed to measure the daunorubicin effect on the membranes' microviscosity. The overall results support that daunorubicin permeation depends on the phospholipid membrane composition and causes alterations in the biophysical properties of the bilayers, namely in the membrane fluidity. The interaction of daunorubicin with the studied phospholipids is mainly driven by electrostatic and hydrophobic interactions. These insights demonstrated that not only membranes can affect daunorubicin accumulation in cells but the compound can alter the properties of membranes. The changes produced by daunorubicin on the lipid structure may constitute an additional mechanism of action, which might lead to modifications in the location and, consequently, the activity of membrane signaling proteins.

Prevalence, incidence, and risk factors of human immunodeficiency virus infection in blood donors in the Southeastern United States.

BACKGROUND: Human immunodeficiency virus (HIV)-positive blood donors pose a risk to blood safety. The Southeastern United States has the highest reported HIV infection rates. Here we calculate HIV prevalence, incidence, and residual risk in Southeastern US blood donors and report risk factors disclosed by incident donors in counseling sessions. STUDY DESIGN AND METHODS: American Red Cross donation and testing data from 2009 to 2014 for three Southeastern collection regions were used to calculate HIV prevalence, incidence, and residual risk. Incident donors had a previous HIV-negative donation within 730 days of their positive donation. Residual risk was defined as the window period multiplied by incidence. RESULTS: From 2009 to 2014, a total of 236 HIV-positive donors occurred in these regions for an overall prevalence of 8.3 per 100,000 donations. There were 56 incident donors over the 6-year period with incidence decreasing from 7.1 per 100,000 person-years (PYs) in the first two years (2009-2010) to 3.5 in the last two years (2013-2014). Residual risk decreased from 1 in 562,000 to 1 in 1,100,000. The most commonly reported risk factor behavior in male incident donors was men who have sex with men; females expressed no predominant risk factor. CONCLUSION: HIV prevalence and incidence among blood donors in the southeast are higher than other US regions, consistent with general public health surveillance. However, the overall residual risk estimates are low at less than 1 per million. Ongoing monitoring of the blood supply along with educational efforts to provide infected individuals with alternatives to donation remain important initiatives.

Solanum diploconos fruits: profile of bioactive compounds and in vitro antioxidant capacity of different parts of the fruit.

Solanum diploconos is an unexploited Brazilian native fruit that belongs to the same genus of important food crops, such as tomato (Solanum lycorpersicum) and potato (Solanum tuberosum). In this study, we determined, for the first time, the profile of bioactive compounds (phenolic compounds, carotenoids, ascorbic acid and tocopherols) of the freeze-dried pulp and peel of Solanum diploconos fruits, as well as of an extract obtained from the whole fruit. Additionally, the antioxidant potential of the whole fruit extract was evaluated in vitro, against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Eighteen phenolic compounds were identified in the peel and pulp and 6 compounds were found in the whole fruit extract. Coumaric, ferulic and caffeic acid derivatives were revealed to be the major phenolic constituents. All-trans-ß-carotene was the major carotenoid (17-38 µg g(-1), dry basis), but all-trans-lutein and 9-cis-ß-carotene were also identified. The peel and pulp presented <2 µg per mL of tocopherols, and ascorbic acid was not detected. The whole fruit extract exhibited scavenging capacity against all tested ROS and RNS (IC50 = 14-461 µg mL(-1)) with high antioxidant efficiency against HOCl. Thus, Solanum diploconos fruits may be seen as a promising source of bioactive compounds with high antioxidant potential against the most physiologically relevant ROS and RNS.

Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 µM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste.