Disposable Amperometric Immunosensor for the Detection of Adulteration in Milk through Single or Multiplexed Determination of Bovine, Ovine, or Caprine Immunoglobulins G.

This paper reports the first immunoplatforms for the detection of adulteration in milk with milk or colostrum from other animals. The developed electrochemical bioplatforms allow the reliable determination of immunoglobulins G (IgGs) from cows, sheeps, or goats. They rely on sandwiching each animal species-specific IgGs with selective antibody pairs [unconjugated and conjugated with horseradish peroxidase (HRP)] onto magnetic microbeads (MBs) used as solid supports and amperometric transduction with the H2O2/hydroquinone (HQ) system at disposable electrodes. The immunoplatforms allow achieving limits of detection (LODs) of 0.74, 0.82, and 0.66 ng mL-1 for bovine, ovine, and caprine IgGs, respectively, which are lower than those obtained with conventional enzyme-linked immunosorbent assay (ELISA) methodologies and in 2-5 times shorter time. The bioplatforms were successfully applied to the determination of the individual content of the target IgGs in milk samples of different animals (cow, sheep, and goat) and type (colostrum, raw, and pasteurized), without matrix effect and after just a sample dilution. They were also applied to the detection of adulteration with milks from other animals at levels below than those required by the European legislation (1.0%, v/v). The possibility to detect milk adulteration with colostrum using a strategy based on the measurement of the total content of the three target IgGs in raw milks is also demonstrated. Multiplexing platforms were constructed to be used in routine surveillance of milk. They are able to provide in a single run and in just 30 min relevant information regarding the milk sample including its animal origin, the undergone heat treatment, and whether it was adulterated with milk or colostrum from other species.

Sensitive ELISA and lateral flow immunoassay for the detection of walnut traces in processed food and working surfaces.

Walnut represents one of the most allergenic nuts that can be found as a hidden allergen. In this study, sandwich ELISA and lateral flow immunoassay (LFIA), based on the determination of Jug r 1, were developed to detect walnut. Cross-reactivity was only found with Pecan nut among a panel of 88 food ingredients tested. ELISA and LFIA could detect 0.25 and 0.5 µg/g of walnut protein in complex food matrices spiked with walnut extract, respectively. Furthermore, walnut was detected in blended (chocolate) and incurred foods (ice cream and bread) added with ground walnut at levels of 0.5 and 1.5 µg protein/g by ELISA and LFIA, respectively. LFIA could also detect 0.1 µg of walnut protein in working surfaces. ELISA displayed acceptable precision and high recovery (71-97 %) and both tests were robust. This study shows that developed ELISA and LFIA are reliable tools to be applied in allergen control programs.

A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs.

Introduction: Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available. Methods: Herein, the first comparative study of six "in house" and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT-qPCR results) as a reference. Four "in house" tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera). Results and discussion: ELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

Cell fate after mitotic arrest in different tumor cells is determined by the balance between slippage and apoptotic threshold.

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x(L) and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x(L) gene was silenced in A549 cells, vincristine induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x(L) or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x(L) switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x(L)/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types.

Development of sandwich ELISA and lateral flow immunoassay to detect almond in processed food.

Almond (Prunus dulcis) represents a potential allergenic hazard that should be included in Allergen Control Plans. In this study, sandwich ELISA and lateral flow immunoassay (LFIA), using amandin (Pru du 6) as the target protein, were developed to detect almond in processed food and validated according to international guides. ELISA could detect 2 ng/mL and LFIA 30 ng/mL of pure amandin. No cross-reactivity was found on a panel of 50 food commodities with the exception of Pecan nut, Brazil nut and chestnut for which the cross-reactivity was lower than 0.02%. Furthermore, ELISA and LFIA were able to detect 0.12 and 0.70 ppm of almond protein in foods spiked with almond extract whereas 0.20 and 2.0 ppm could be detected in baked cookies incurred with almond, respectively. Both techniques could be applied for food manufacturers and control agencies for monitoring the presence of almond traces in food and working surfaces.

Ultrasensitive detection of soy traces by immunosensing of glycinin and ß-conglycinin at disposable electrochemical platforms.

This work reports the first electrochemical bioplatform for the determination of soy traces in food. The bioplatform involves sandwich-type immunoassays using specific antibodies for ß-conglycinin and glycinin, which are the main allergenic soy proteins, and carboxylic acid-modified magnetic microbeads. Amperometric detection at -0.20 V (vs. an Ag pseudo-reference electrode) was performed using single or dual screen-printed carbon electrodes and the H2O2/hydroquinone (HQ) system. The measured variation in the cathodic current was directly proportional to the concentration of target allergenic proteins. The developed bioplatforms exhibit a good selectivity and sensitivity providing limits of detection (LOD) values of 0.03 and 0.02 ng mL-1 for ß-conglycinin and glycinin, respectively. The determination of both proteins can be carried out in only 1.5 h. The electrochemical bioplatforms allow their accurate determinations (with results statistically comparable to those provided by ELISA methodologies) in raw cookie dough and baked cookies enriched with soy flour. The results obtained confirm, in a pioneering way with electrochemical biosensors, the possibility of discriminating samples incurred with as little as 0.0005 ppm of a food allergen in model cookie extracts.

Bim is the key mediator of glucocorticoid-induced apoptosis and of its potentiation by rapamycin in human myeloma cells.

Glucocorticoids are widely used in anti-myeloma therapy and their action is potentiated by rapamycin, a mTOR inhibitor. However, the molecular mechanisms underlying these effects remain poorly characterized. We show here that dexamethasone (Dex)-induced apoptosis in MM.1S and OPM-2 cells is characterized by Bax and Bak conformational changes, DeltaPsi(m) loss, cytochrome c release and caspase-3 activation. Rapamycin, which had minimal cytotoxic effect by itself, strongly potentiated Dex-induced apoptosis. Apoptotic gene expression profiling showed an increase in mRNA levels of Bim in MM.1S cells after Dex treatment and further increases in both cell lines when co-treated with rapamycin. Western blot analysis revealed a moderate increase in Bim protein levels in both MM.1S and OPM-2 cells. Immunoprecipitation experiments revealed that most Bim was complexed to Mcl-1 in untreated cells. Upon treatment with Dex, and specially Dex plus rapamycin, Bim-Mcl-1 complex was disrupted and Bim was found associated to a CHAPS-insoluble fraction. Overexpression of Mcl-1 stabilized Bim-Mcl-1 complexes upon treatment with Dex or Dex+rapamycin and fully prevented apoptosis. Gene silencing of Bim inhibited for the most part Dex-induced apoptosis and, to a large extent, apoptosis induced by Dex plus rapamycin. These results, taken together, indicate that Bim protein is the key mediator of apoptosis induced by Dex and also responsible for the potentiating effect of rapamycin, providing molecular criteria for the use of glucocorticoids combined with mTOR inhibitors in myeloma therapy.

AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics.

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.

Influence of different extraction conditions on the detection of glycinin and ß-conglycinin in model processed foods by ELISA.

The presence of undeclared soy proteins in food can cause severe reactions in soy allergic individuals. The extraction of target proteins from processed foods is a crucial step in allergen detection by immunoassays, as only successfully extracted target proteins can be detected by the specific antibodies. The effectiveness was studied of different conditions (type of buffer, temperature and time of incubation) on the extraction of total protein, and concentration of glycinin and ß-conglycinin from different food matrices. The yields were determined using a soy protein isolate and three processed foods (sausage, bread and pâté) incurred with soy proteins. The yields were affected by the processing of analysed products and the composition and pH of the extraction buffers. Neutral and alkaline buffers (pH from 7.4 to 10.6) exhibited good protein extraction capacity and detectability of the specific target proteins. Denaturing additives and highly alkaline buffer (pH 12) extracted more crude protein but they were incompatible with the ELISA assay. Overall, the best results were obtained using phosphate (pH 7.4) and Tris/HCl (pH 8.5) buffers in the presence of 0.5 M NaCl. Crude protein yield of food extracts did not correlate with that of glycinin and ß-conglycinin, whereas a good relationship was found between the yields of the two proteins.

Development of a novel duplex lateral flow test for simultaneous detection of casein and ß-lactoglobulin in food.

Milk by-products such as whey and caseinate are widely used as ingredients or processing aids in food industry. However, since they could cause allergic reactions they are included in Allergen Control Plans. ß-Lactoglobulin is the major whey protein and caseins are main proteins in milk. Selection of a unique target to analyze the presence of milk in foods could be insufficient when the source of milk proteins is unknown. A new test based on lateral flow immunocromatography that combines the simultaneous and independent detection of both proteins (ß-lactoglobulin and casein) in one rapid test was developed. The assay was validated according to AOAC guidelines being able to detect ß-lactoglobulin (0.5 ppm), casein (2 ppm), whey and powder milk (1-5 ppm). No cross-reactivity was found with a panel of 38 food commodities. The method is a rapid and suitable tool to identify milk proteins in processed food, ingredients, and rinsing water.

Validation of a rapid lateral flow method for the detection of cows' milk in water buffalo, sheep or goat milk.

For many years, the adulteration of milk from sheep, goats or water buffalos with cows' milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows' milk in milk from other species, including buffalo's milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows' milk. This level is below the current EU allowed level of cows' milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows' milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.

Stakeholders' Guidance Document for Consumer Analytical Devices with a Focus on Gluten and Food Allergens.

Until recently, analytical tests for food were performed primarily in laboratories, but technical developments now enable consumers to use devices to test their food at home or when dining out. Current consumer devices for food can determine nutritional values, freshness, and, most recently, the presence of food allergens and substances that cause food intolerances. The demand for such products is driven by an increase in the incidence of food allergies, as well as consumer desire for more information about what is in their food. The number and complexity of food matrixes creates an important need for properly validated testing devices with comprehensive user instructions (definitions of technical terms can be found in ISO 5725-1:1994 and the International Vocabulary of Metrology). This is especially important with food allergen determinations that can have life-threatening consequences. Stakeholders-including food regulators, food producers, and food testing kit and equipment manufacturers, as well as representatives from consumer advocacy groups-have worked to outline voluntary guidelines for consumer food allergen- and gluten-testing devices. These guidelines cover areas such as kit validation, user sampling instructions, kit performance, and interpretation of results. The recommendations are based on (1) current known technologies, (2) analytical expertise, and (3) standardized AOAC INTERNATIONAL allergen community guidance and best practices on the analysis of food allergens and gluten. The present guidance document is the first in a series of papers intended to provide general guidelines applicable to consumer devices for all food analytes. Future publications will give specific guidance and validation protocols for devices designed to detect individual allergens and gluten, as statistical analysis and review of any validation data, preferably from an independent third party, are necessary to establish a device's fitness-for-purpose. Following the recommendations of these guidance documents will help ensure that consumers are equipped with sufficient information to make an informed decision based on an analytical result from a consumer device. However, the present guidance document emphasizes that consumer devices should not be used in isolation to make a determination as to whether a food is safe to eat. As advances are made in science and technology, these recommendations will be reevaluated and revised as appropriate.

Granulysin induces apoptotic cell death and cleavage of the autophagy regulator Atg5 in human hematological tumors.

Granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work demonstrated that granulysin caused cell death through mitochondrial damage with release of AIF and cytochrome c. However, the molecular mechanism and, especially, the type of cell death were still not well defined. In the present work we show that granulysin-induced cell death is apoptotic, with phosphatidylserine exposure preceding membrane breakdown and with caspase 3 activation. Granulysin-induced apoptosis is prevented in Jurkat cells over-expressing Bcl-xL or Bcl2, or lacking Bak and Bax or Bim expression, suggesting a central role of the mitochondrial apoptotic pathway. This apoptotic process is initiated by intracellular Ca(2+) increase and mitochondrial ROS generation. We have tested granulysin against other hematological tumor cells such as multiple myeloma cell lines, and cells from B cell chronic lymphocytic leukemia (B-CLL) patients, finding different degrees of sensitivity. We also show that granulysin induces the cleavage of Atg5 in the complex formed with Atg12, without affecting autophagy. In conclusion, granulysin induces apoptosis on hematological tumor cells and on cells from B-CLL patients, opening the door to research on its use as a new anti-tumoral treatment.

Bortezomib resistance in a myeloma cell line is associated to PSMß5 overexpression and polyploidy.

Bortezomib is a proteasome inhibitor important to the therapy of multiple myeloma (MM), though a number of patients show resistance to this drug. To study the cellular basis of this resistance we have generated a MM cell line displaying enhanced (5-6-fold) resistance to bortezomib by serial cultivation of RPMI 8226 cells with increasing concentrations of this drug. Bortezomib-resistant cells (8226/7B) became bigger in size than parental cells and nearly doubled the amount of DNA per cell, evolving from hypotriploidy to near-tetraploidy. 8226/7B displayed lowered Noxa accumulation and reduced caspase-3 activation in response to bortezomib. Resistant 8226/7B cells overexpressed the PSMß5 proteasome subunit, the molecular target of bortezomib, both at the mRNA and protein level. No mutations were detected in the PSMß5 gene. Bortezomib-resistant cells were roughly as sensitive as parental cells to other chemotherapeutic drugs, including doxorubicin, melphalan, vincristine, BMS-214662 and BMS-345541. 8226/7B cells showed partial and high cross-resistance to the proteasome inhibitors epoxomicin and MG-132, respectively. Co-treatment with the histone deacetylase inhibitor trichostatin A (TSA) potentiated bortezomib-induced apoptosis in parental RPMI 8226 cells but did not revert bortezomib resistance in 8226/7B cells. Therefore, treatment of bortezomib-refractory myeloma with drugs targeting molecular structures other than proteasome seems to be the more suitable therapeutic strategy to overcome bortezomib resistance.

Different contribution of BH3-only proteins and caspases to doxorubicin-induced apoptosis in p53-deficient leukemia cells.

Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway, either facilitating (Bax, Bak, BH3-only) or inhibiting (Bcl-2, Bcl-x(L), Mcl-1, A1) mitochondrial release of apoptogenic factors. The role of caspases in this process is a matter of controversy. We have analyzed the relative contribution of caspases and Bcl-2 family of proteins in the induction phase of apoptosis triggered by doxorubicin in two p53-deficient leukemia cell lines, Jurkat and U937. First, we have found that caspases are dispensable for the induction phase of doxorubicin-induced apoptosis in both cell lines but they are needed to speed up the execution phase in Jurkat cells, not expressing Bax. Thus, down-regulation of Bak expression by siRNA significantly prevented doxorubicin-induced apoptosis in Jurkat but not in U937 cells. Reduction of Mcl-1 protein levels with siRNA increased sensitivity to apoptosis in both cell lines. Moreover, our results indicate that the contribution of BH3-only proteins to apoptosis is cell line specific. In Jurkat cells simultaneous silencing of Bim and PUMA was necessary to reduce doxorubicin-induced apoptosis. In U937 cells silencing of Bim or Noxa reduced sensitivity to doxorubicin. Immunoprecipitation experiments discarded an interaction between Mcl-1 and Bak in both cell lines and underscored the role of Bim and PUMA as mediators of Bax/Bak activation.

Cooperation between Apo2L/TRAIL and bortezomib in multiple myeloma apoptosis.

The proteasome inhibitor bortezomib is currently an important drug for treatment of relapsed and refractory multiple myeloma (MM) and for elderly patients. However, cells from some patients show resistance to bortezomib. We have evaluated the possibility of improving bortezomib therapy with Apo2L/TRAIL, a death ligand that induces apoptosis in MM but not in normal cells. Results indicate that cotreatment with low doses of bortezomib significantly increased apoptosis of MM cells showing partial sensitivity to Apo2L/TRAIL. Bortezomib treatment did not significantly alter plasma membrane amount of DR4 and DR5 but increased Apo2L/TRAIL-induced caspase-8 and caspase-3 activation. Apo2L/TRAIL reverted bortezomib-induced up-regulation of beta-catenin, Mcl-1 and FLIP, associated with the enhanced cytotoxicity of combined treatment. More important, some cell lines displaying resistance to bortezomib were sensitive to Apo2L/TRAIL-induced apoptosis. A cell line made resistant by continuous culture of RPMI 8226 cells in the presence of bortezomib (8226/7B) was highly sensitive to Apo2L/TRAIL-induced apoptosis. Moreover, RPMI 8226 cells overexpressing Mcl-1 (8226/Mcl-1) or Bcl-x(L) (8226/Bcl-x(L)) also showed enhanced resistance to bortezomib, but co-treatment with Apo2L/TRAIL reverted this resistance. These results indicate that Apo2L/TRAIL can cooperate with bortezomib to induce apoptosis in myeloma cells and can be an useful adjunct for MM therapy.