Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 µL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.

Antiviral action of aqueous extracts of propolis from Scaptotrigona aff. postica (Hymenoptera; Apidae) against Zica, Chikungunya, and Mayaro virus.

The limited availability of antivirals for new highly pathogenic strains of virus has become a serious public health. Therefore, news products against these pathogens has become an urgent necessity. Among the multiple sources for news antibiotics and antivirals, insect exudates or their products has become an increasingly frequent option. Insects emerged 350 million years ago and have showed a high adaptability and resistance to the most varied biomes. Their survival for so long, in such different environments, is an indication that they have a very efficient protection against environmental infections, despite not having a developed immune system like mammals. Since the ancient civilizations, the products obtained from the bee have been of great pharmacological importance, being used as antimicrobial, anti-inflammatory, antitumor and several other functions. Investigations of biological activity of propolis have been carried out, mainly in the species Apis mellifera, and its product have showed activity against some important viruses. However, for the Meliponini species, known as stingless bees, there are few studies, either on their chemical composition or on their biological activities. The importance of studying these bees is because they come from regions with native forests, and therefore with many species of plants not yet studied, in addition to which they are regions still free of pesticides, which guarantees a greater fidelity of the obtained data. Previous studies by our group with crude hydroalcoholic extract of propolis demonstrated an intense antiviral activity against Herpes, influenza, and rubella viruses. In this work, we chose to use aqueous extracts, which eliminates the presence of other compounds besides those originally present in propolis, in addition to extracting substances different from those obtained in alcoholic extracts. Therefore, this study aimed to identify, isolate and characterize compounds with antiviral effects from aqueous propolis extracts from Scaptotrigona aff postica, in emerging viruses such as zicavirus, chikungunya, and mayaro virus. The evaluation of the antiviral activity of the crude and purified material was performed by reducing infectious foci in VERO cell cultures. The results obtained with crude propolis, indicate a high reduction of zica virus (64×) and mayaro (128×) when was used 10% v/v of propolis. The reduction of chikungunya virus was of 256 fold, even when was used 5% v/v of propolis. The chemical characterization of the compounds present in the extracts was performed by high-pressure liquid chromatography. Through the purification of propolis by HPLC and mass spectrometry, it was possible to identify and isolate a peak with antiviral activity. This substance showed activity against all viruses tested. When purified fraction was used, the reduction observed was of 16 fold for zicavirus, 32 fold for mayaro virus and 512 fold for chikungunya virus. Likewise, it was observed that the antiviral response was concentration dependent, being more intense when propolis was added 2 h after the viral infection. Now we are carrying out the chemical characterization of the purified compounds that showed antiviral action.

Effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on NRF2, KEAP1, SOD1, and GPX2 expression in human colorectal cancer cell lines.

We investigated the effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on the expression of cytoprotective genes nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2), kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase (SOD1), and glutathione peroxidase (GPX2) in human colorectal cancer cell lines (HT-29 and Caco-2). Cells were cultured for 24 h in Dulbecco's Modified Eagle's Medium containing juçara fruit pulp (5, 10, or 50 mg/mL) or lyophilized extract (0.05, 0.1, or 0.5 mg/mL), and gene expression was quantified using real-time quantitative reverse transcription polymerase chain reaction. All studied genes showed significant variation in gene expression among different concentrations of pulp or lyophilized extract. Overall, the expression of the selected genes decreased in both cell lines following exposure to the pulp or lyophilized extract in a dose-dependent manner for most of the concentrations studied. In summary, our study showed that the compounds in juçara fruit inhibited the expression of cytoprotective genes associated with the antioxidant response and that, although not cytotoxic at the concentrations studied, they could potentially block the activation of the NRF2/KEAP1 pathway.

Effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on NRF2, KEAP1, SOD1, and GPX2 expression in human colorectal cancer cell lines

We investigated the effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on the expression of cytoprotective genes nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2), kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase (SOD1), and glutathione peroxidase (GPX2) in human colorectal cancer cell lines (HT-29 and Caco-2). Cells were cultured for 24 h in Dulbecco's Modified Eagle's Medium containing juçara fruit pulp (5, 10, or 50 mg/mL) or lyophilized extract (0.05, 0.1, or 0.5 mg/mL), and gene expression was quantified using real-time quantitative reverse transcription polymerase chain reaction. All studied genes showed significant variation in gene expression among different concentrations of pulp or lyophilized extract. Overall, the expression of the selected genes decreased in both cell lines following exposure to the pulp or lyophilized extract in a dose-dependent manner for most of the concentrations studied. In summary, our study showed that the compounds in juçara fruit inhibited the expression of cytoprotective genes associated with the antioxidant response and that, although not cytotoxic at the concentrations studied, they could potentially block the activation of the NRF2/KEAP1 pathway.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 µm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab of antibodies on the beads. Different patterns of digestion rates were found due to proteolysis of the oriented and nonoriented antibodies on MBs. For oriented antibodies, the peptides from outer Fab regions gave a much higher digestion rate than those from Fc regions, while for randomly immobilized antibodies digestion rates for Fab and Fc peptides were similar. This novel approach is a useful and convenient tool to characterize antibody orientation for immunoassays and other applications. The relative degree of orientation can be assessed using a metric Ro denoting amount of Fab marker peptides found divided by Fc + Fab marker peptides × 100%. Oriented antibodies on the MBs also provided more efficient antigen capture compared to randomly immobilized antibodies.

Lacrain: the first antimicrobial peptide from the body extract of the Brazilian centipede Scolopendra viridicornis.

Antimicrobial activities have previously been described by traditional Eastern medicine in Chilopoda body extracts, but until now no bioactive peptides have been described. In this study, a novel antimicrobial peptide, lacrain, was isolated from the body extract of the Brazilian Chilopoda Scolopendra viridicornis. The peptide was isolated by reverse-phase high-performance liquid chromatography (RP-HPLC). Its activity was tested using a liquid growth inhibition assay and the peptide was characterised using mass spectrometry. Lacrain has a sequence composed of eight amino acid residues and a molecular mass of 925.5 Da. A synthetic peptide of the native lacrain had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against Gram-negative bacteria, showing strong bactericidal activity. Moreover, the peptide did not present haemolytic activity against human erythrocytes. Lacrain represents a novel molecule with powerful antibacterial activity that could be used as a new template for the development of drugs against clinically resistant bacterial strains.

The relationship between emergence from spawning gravel and growth in farmed rainbow trout Oncorhynchus mykiss.

The relationship between the timing of emergence from spawning gravel and growth after emergence was investigated in farmed Oncorhynchus mykiss. A relationship between the time of emergence and growth became evident after 6 months of rearing, where individuals with an intermediate emergence time had grown larger compared with early and late emerging individuals.

Rondonin an antifungal peptide from spider (Acanthoscurria rondoniae) haemolymph.

Antimicrobial activities were detected in the haemolymph of the spider Acanthoscurrria rondoniae. A novel antifungal peptide, rondonin, was purified by reverse phase high performance liquid chromatography (RP-HPLC). Rondonin has an amino acid sequence of IIIQYEGHKH and a molecular mass of 1236.776 Da. This peptide has identity to a C-terminal fragment of the "d" subunit of haemocyanin from the spiders Eurypelma californicum and Acanthoscurria gomesiana. A synthetic peptide mimicking rondonin had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against fungus. These data led us to conclude that the antifungal activity detected in the plasma of these spiders is the result of enzymatic processing of a protein that delivers oxygen in the haemolymph of many chelicerate. Several studies have suggested that haemocyanins are involved in the arthropod immune system, and the activity of this haemocyanin fragment reinforces this idea.

Effects of maternal stress coping style on offspring characteristics in rainbow trout (Oncorhynchus mykiss).

Maternal size, age, and allostatic load influence offspring size, development, and survival. Some of these effects have been attributed to the release of glucocorticoids, and individual variation in these stress hormones is related to a number of traits. Correlated traits are often clustered and used to define the proactive and reactive stress coping styles. Although stress coping styles have been identified in a number of animal groups, little is known about the coupling between stress coping style and offspring characteristics. In the present study, plasma cortisol levels in ovulated mothers and cortisol levels in non-fertilized eggs from two rainbow trout (Oncorhynchus mykiss) strains selected for high (HR) and low (LR) post-stress plasma cortisol levels were compared. Offspring characteristics such as egg size, larval growth, and energy reserves also were compared between the two strains. Maternal plasma and egg cortisol levels were correlated, but no difference between the HR and LR strains was detected in either parameter. LR females produced larger eggs, and larvae with larger yolk sacs compared to HR females, however no differences in larval body size (excluding the yolk) was detected between strains. Considering that the HR and LR strains have a number of correlated behavioral and physiological traits that resemble the reactive and proactive stress coping styles, respectively, the results suggest that proactive mothers invest more energy into their offspring, producing larvae with larger energy reserves. It is possible that larger energy reserves in proactive larvae support the energy requirement for establishing and defending territory in salmonid fish. Furthermore, in the present study we found a positive relationship between mother plasma cortisol and egg cortisol; however neither mother plasma cortisol nor egg cortisol differed between strains. These results indicate that cortisol endowment from the mother to the offspring plays a minor role in the transfer of the behavioral and physiological traits which separates these strains.