rgbradford-protein-quantitation-with-a-smartphone-camera

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H] = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra).

This study aims to isolate metal-binding peptides and synthesize promising amino acid sequences to potentially act as neuroprotective compounds in the future, targeting different mechanisms. Fractions of whey metal-binding peptides (Cu, Fe, and Zn) isolated by immobilized metal affinity chromatography showed different amino acid profiles according to the metal. The Cu-binding peptides presented roughly twofold increase in the  antioxidant, as assessed by oxygen radical absorbance capacity and anticholinesterase activities over the hydrolysate. This is probably because of the higher concentration of aromatic and basic residues, the latter being crucial for binding to the anionic sites of acetylcholinesterase. Six peptide sequences were synthesized based on the metal-binding sites, molecular mass, hydrophobicity, and bioactivity probability. Among the synthetic peptides, the VF dipeptide stood out both for its  antioxidant and anticholinesterase activities. This peptide, as well as the fraction of Cu-binding peptides, should be further studied because it may act through different mechanisms related to neurodegenerative diseases, in addition to the chelation of the excess of metals in the central nervous system.

Isolation and Sequencing of Cu-, Fe-, and Zn-Binding Whey Peptides for Potential Neuroprotective Applications as Multitargeted Compounds.

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of  () . Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper ().

Metabolic diseases are increasing at staggering rates globally. The peroxisome proliferator-activated receptors (PPARα/γ/δ) are fatty acid sensors that help mitigate imbalances between energy uptake and utilization. Herein, we report compounds derived from phenolic lipids present in cashew nut shell liquid (CNSL), an abundant waste byproduct, in an effort to create effective, accessible, and sustainable drugs. Derivatives of anacardic acid and cardanol were tested for PPAR activity in HEK293 cell co-transfection assays, primary hepatocytes, and 3T3-L1 adipocytes.  studies using PPAR-expressing zebrafish embryos identified CNSL derivatives with varying tissue-specific activities. LDT409 () is an analogue of cardanol with partial agonist activity for PPARα and PPARγ. Pharmacokinetic profiling showed that  is orally bioavailable with a half-life of 4 h in mice. CNSL derivatives represent a sustainable source of selective PPAR modulators with balanced intermediate affinities (EC ∼ 100 nM to 10 μM) that provide distinct and favorable gene activation profiles for the treatment of diabetes and obesity.

Phenolic Lipids Derived from Cashew Nut Shell Liquid to Treat Metabolic Diseases.

 is used to prepare the psychoactive beverage ayahuasca, and both have therapeutic potential for the treatment of many central nervous system (CNS) conditions. This study aimed to isolate new bioactive compounds from  extract and evaluate their biological activity, and that of the known β-carboline components of the plant (harmine, harmaline, and tetrahydroharmine), in BV-2 microglial cells, the in vivo activation of which is implicated in the physiopathology of CNS disorders.  extract was fractionated using semipreparative liquid chromatography (HPLC-DAD) and the exact masses ([M + H]/) of the compounds in the 5 isolated fractions were determined by high-resolution LC-MS/MS: F1 (174.0918 and 233.1289), F2 (353.1722), F3 (304.3001), F4 (188.1081), and F5 (205.0785). Harmine (75.5-302 µM) significantly decreased cell viability after 2 h of treatment and increased the number of necrotic cells and production of reactive oxygen species at equal or lower concentrations after 24 h. F4 did not impact viability but was also cytotoxic after 24 h. Most treatments reduced proinflammatory cytokine production (IL-2, IL-6, IL-17, and/or TNF), especially harmaline and F5 at 2.5 µM and higher concentrations, tetrahydroharmine (9.3 µM and higher), and F5 (10.7 µM and higher). The results suggest that the compounds found in  extract have anti-inflammatory potential that could be explored for the development of treatments for neurodegenerative diseases.

Components of , a Plant Used in the Preparation of the Psychoactive Ayahuasca, Induce Anti-Inflammatory Effects in Microglial Cells.

Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.

Neuroprotective effects on microglia and insights into the structure-activity relationship of an antioxidant peptide isolated from Pelophylax perezi.

Editorial: Redox Metabolism in Environmental and Ecological Physiology of Animals.

The color shift caused by the interaction between proteins and Coomassie Brilliant Blue in the Bradford assay can be recorded with a smartphone camera. Color data can then be extracted from pictures and processed as analytical signals. Here, I present a simple procedure to accurately measure protein levels using color data from pictures of microplates. Plotting the ratio of blue to green intensity (RGB scale) as a function of protein concentration results in a linear (R ≥ 0.99) relationship, from which protein levels in biological samples can be calculated with no significant difference from values obtained using absorbance data (RGB versus absorbance curves).

RGBradford: Accurate measurement of protein concentration using a smartphone camera and the blue to green intensity ratio.

This commentary acknowledges the contributions of the Ukrainian biologist, Dr. Volodymyr Lushchak, to the understanding of the physiological adaptive strategy called "Preparation for Oxidative Stress" (POS). In the 1990s, various studies revealed that activities of antioxidant enzymes rose in animals under hypometabolic conditions. These timely observations allowed scientists to propose that this increase could prepare animals for reoxygenation events following the release of oxygen restriction, but in doing so, would trigger oxidative damage, hence the use of the term "preparation". Over next 25 years, the phenomenon was described in detail in more than one hundred studies of animals under conditions of aestivation, hypoxia/anoxia, freezing, severe dehydration, ultraviolet exposure, air exposure of water-breathing animals, salinity stress, and others. The POS phenomenon remained without a mechanistic explanation until 2013, when it was proposed that a small increase in oxyradical formation during hypoxia exposure (in hypoxia-tolerant animals) could activate redox-sensitive transcription factors that, in turn, would initiate transcription and translation of antioxidant enzymes. Dr. Lushchak, who studied goldfish under severe hypoxia in the 1990s, had actually proposed the increased production of oxyradicals under this condition and concluded that it would lead to an upregulation of antioxidant enzymes, the hallmark of the POS strategy. However, his research partner at the time, Dr. Hermes-Lima, thought the idea did not have sufficient evidence to support it and recommended the removal of this explanation. In those days, the main line of thinking was that increased oxyradical formation under hypoxia was "impossible". So, as it turns out, the ideas of Dr. Lushchak were well ahead of his time. It then took >10 years before the biochemical and molecular mechanisms responsible for triggering the POS response were clarified. In the present article, this fascinating history is described to highlight Dr. Lushchak's contributions and insights about the POS theory.

Commentary: On the merit of an early contributor of the "Preparation for Oxidative Stress" (POS) theory.

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake . This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom ().

Preparation for oxidative stress (POS) is a widespread adaptive response to harsh environmental conditions, whose hallmark is the upregulation of antioxidants. In contrast to controlled laboratory settings, animals are exposed to multiple abiotic stressors under natural field conditions. Still, the interplay between different environmental factors in modulating redox metabolism in natural settings remains largely unexplored. Here, we aim to shed light on this topic by assessing changes in redox metabolism in the mussel  naturally exposed to a tidal cycle. We compared the redox biochemical response of mussels under six different natural conditions in the field along two consecutive days. These conditions differ in terms of chronology, immersion/emersion, and solar radiation, but not in terms of temperature. Animals were collected after being exposed to air early morning (7:30), immersed during late morning and afternoon (8:45-15:30), and then exposed to air again late afternoon towards evening (17:45-21:25), in two days. Whole body homogenates were used to measure the activity of antioxidant (catalase, glutathione transferase and glutathione reductase) and metabolic (glucose 6-phosphate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and pyruvate kinase) enzymes, reduced (GSH) and disulfide (GSSG) glutathione levels, and oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances). Air and water temperature remained stable between 22.5 °C and 26 °C during both days. Global solar radiation (GSR) greatly differed between days, with a cumulative GSR of 15,381 kJ/m for day 1 and 5,489 kJ/m for day 2, whose peaks were 2,240 kJ/m/h at 14:00 on day 1 and 952 kJ/m/h at 12:00 on day 2. Compared with animals underwater, emersion during early morning did not elicit any alteration in redox biomarkers in both days. Air exposure for 4 h in the late afternoon towards evening caused oxidative damage to proteins and lipids and elicited GSH synthesis in animals that had been previously exposed to high GSR during the day. In the following day, when GSR was much lower, exposure to air under the same conditions (duration, time, and temperature) had no effect on any redox biomarker. These findings suggest that air exposure under low-intensity solar radiation is not sufficient to trigger POS in  in its natural habitat. Thus, natural UV radiation is possibly a key environmental factor that combined to air exposure induces the POS-response to the stressful event of tidal variation in this coastal species.

The role of solar radiation and tidal emersion on oxidative stress and glutathione synthesis in mussels exposed to air.

Editorial: The defense responses of aquatic animals to the environment.

The upregulation of endogenous antioxidants is a widespread phenomenon in animals that tolerate hypoxia/anoxia for extended periods. The identity of the mobilized antioxidant is often context-dependent and differs among species, tissues, and stresses. Thus, the contribution of individual antioxidants to the adaptation to oxygen deprivation remains elusive. This study investigated the role of glutathione (GSH) in the control of redox homeostasis under the stress of anoxia and reoxygenation in , an animal model of anoxia tolerance. To do so, the total GSH (tGSH) pool was depleted with l-buthionine-(S, R)-sulfoximine (BSO) before exposing snails to anoxia for 6 h. Then, the concentration of GSH, glutathione disulfide (GSSG), and oxidative stress markers (TBARS and protein carbonyl) and the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione transferase, glutathione reductase, and glucose 6-phosphate dehydrogenase) were measured in foot muscle and hepatopancreas. BSO alone induced tGSH depletion by 59-75%, but no other changes happened in other variables, except for foot GSSG. Anoxia elicited a 110-114% increase in glutathione peroxidase in the foot; no other changes occurred during anoxia. However, GSH depletion before anoxia increased the GSSG/tGSH ratio by 84-90% in both tissues, which returned to baseline levels during reoxygenation. Our findings indicate that glutathione is required to withstand the oxidative challenge induced by hypoxia and reoxygenation in land snails.

Glutathione Depletion Disrupts Redox Homeostasis in an Anoxia-Tolerant Invertebrate.

Amphibian secretions have been extensively investigated for the production of bioactive molecules. Salamandrin-I is an antioxidant peptide, isolated from the skin secretion of the fire salamander, that has induced no toxicity in microglia or erythrocytes. Importantly, the administration of antioxidants may constitute an adequate therapeutic approach to cancer treatment. Here, with the purpose of better characterizing the therapeutic potential of salamandrin-I, we investigated whether this antioxidant peptide also exerts anticancer activity, using the human leukemia cell line HL-60 as a cancer model. Salamandrin-I treatment induced a significant reduction in HL-60 proliferation, which was accompanied by cell cycle arrest. Furthermore, the peptide-induced cell death showed a significant increase in the LDH release in HL-60 cells. The cellular toxicity exerted by salamandrin-I is possibly related to pyroptosis, since the HL-60 cells showed loss of mitochondrial membrane potential and hyperexpression of inflammasome components following the peptide treatment. This is the first demonstration of the anticancer potential of the salamandrin-I peptide. Such results are important, as they offer relevant insights into the field of cancer therapy and allow the design of future bioactive molecules using salamandrin-I as a template.

Article	Year
The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra). Biomolecules\| PubMed ID: 32230960	2020
Isolation and Sequencing of Cu-, Fe-, and Zn-Binding Whey Peptides for Potential Neuroprotective Applications as Multitargeted Compounds. Journal of agricultural and food chemistry\| PubMed ID: 33095576	2020
Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (). Journal of natural products\| PubMed ID: 34077221	2021
Phenolic Lipids Derived from Cashew Nut Shell Liquid to Treat Metabolic Diseases. Journal of medicinal chemistry\| PubMed ID: 35089724	2022
Components of , a Plant Used in the Preparation of the Psychoactive Ayahuasca, Induce Anti-Inflammatory Effects in Microglial Cells. Molecules (Basel, Switzerland)\| PubMed ID: 35458698	2022
Neuroprotective effects on microglia and insights into the structure-activity relationship of an antioxidant peptide isolated from Pelophylax perezi. Journal of cellular and molecular medicine\| PubMed ID: 35460166	2022
Editorial: Redox Metabolism in Environmental and Ecological Physiology of Animals. Frontiers in physiology\| PubMed ID: 35547571	2022
RGBradford: Accurate measurement of protein concentration using a smartphone camera and the blue to green intensity ratio. Analytical biochemistry\| PubMed ID: 35987416	2022
Commentary: On the merit of an early contributor of the "Preparation for Oxidative Stress" (POS) theory. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology\| PubMed ID: 36368609	2023
Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (). Journal of natural products\| PubMed ID: 36508333	2022
The role of solar radiation and tidal emersion on oxidative stress and glutathione synthesis in mussels exposed to air. PeerJ\| PubMed ID: 37193036	2023
Editorial: The defense responses of aquatic animals to the environment. Frontiers in physiology\| PubMed ID: 37362435	2023
Glutathione Depletion Disrupts Redox Homeostasis in an Anoxia-Tolerant Invertebrate. Antioxidants (Basel, Switzerland)\| PubMed ID: 37371926	2023
The Peptide Salamandrin-I Modulates Components Involved in Pyroptosis and Induces Cell Death in Human Leukemia Cell Line HL-60. Pharmaceutics\| PubMed ID: 37514049	2023

Daniel C. Moreira

Biography

JoVE Journal Publications

Other Publications