Development of sandwich and competitive ELISA formats to determine ß-conglycinin: Evaluation of their performance to detect soy in processed food

Two ELISA formats (sandwich and indirect competitive) were developed to quantify ß-conglycinin, a major soy allergen. Their performance was evaluated using three model foods incurred with soy proteins. The sandwich format detects the addition of 0.005% and 0.05% soy proteins in pasteurized sausages and baked bread. However, the competitive format detects only 0.1 and 0.5%, respectively. ß-conglycinin was not detected in sterilized pâtè with any format. An industrial prototype of the sandwich ELISA was in-house validated, showing acceptable results of repeatability, reproducibility and robustness. Model foods spiked with ß-conglycinin after processing showed recoveries between 93.3 and 138.7%. However, in model foods incurred with soy proteins before processing the recovery decreased with the increase of the severity of heat treatment applied. The sandwich format could differentiate most of the retail foods with soy declared or not as ingredient. The ELISA format and processing conditions greatly influence the determination of ß-conglycinin in food

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. © 2021 The Author(s

Influence of Age on Stress Responses to Metabolic Cage Housing in Rats

SUMMARY 1. We studied the effect of isolation stress in 3-and 12-month-old rats individually housed in metabolic cages for 7 days. Urine (24 hr) was collected daily from one group of animals of each age. The other group was tested in an open field and on a hot plate on days 1 and 7. 2. Total deambulation in the open-field test was lower in young than in older rats both on day 1 (54.7 Ϯ 9.9 vs 80 Ϯ 8.9 crossings/session; P Ͻ 0.04) and on day 7 (21 Ϯ 9 vs 48 Ϯ 7 crossings per session; P Ͻ 0.04) and decreased significantly in the two groups when tested on day 7 (P Ͻ 0.03). Latency to paw-licking in the hot-plate test was longer in young than in older animals on day 1 (14 Ϯ 2 vs 8 Ϯ 4 sec; P Ͻ 0.05) but was similar in the two groups on day 7. 3. Urinary excretions of norepinephrine (NE) and epinephrine (E) were determined by HPLC with electrochemical detection. Urinary NE in day 1 was similar in young and older animals (2627 Ϯ 828 vs 3069 Ϯ 598 ng/24 hr). In young animals NE excretion decreased along the study and was significantly (P Ͻ 0.02) lower than on day 1 during the last 3 days of the study. Conversely, in older animals urinary excretion of NE remained similar throughout the study. On day 7 urinary excretion of NE in older animals was about two fold that in young rats. Urinary E was similar in young and older rats (341 Ϯ 127 vs 532 Ϯ 256 ng/24 hr) on day 1 and showed a tendency to increase throughout the study. 4. Urinary monoamine oxidase inhibitory (IMAO) activity was determined by testing the ability of urine extracts to inhibit rat liver MAO activity in vitro and was higher in young than in older animals throughout the study (day 1, 54.8 Ϯ 4.2 vs 25.1 Ϯ 5.1%; P Ͻ 0.02). In young rats excretion of IMAO was significantly higher during the last 3 days of the study than on day 1 (P Ͻ 0.05). In older animals urinary IMAO showed a tendency to increase at the end of the study. 5. Isolation stress caused by housing rats in metabolic cages results in different behavioral and metabolic responses in young and older animals. Young animals exhibit a lower locomotor and analgesic response and excrete lower amounts of NE and higher IMAO activity in the urine than older rats. The metabolic and behavioral responses to isolation stress are highly dependent on the age of the animals tested. These results should be taken into consideration when designing experiments requiring the use of metabolic cages

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone

Elevated plasma succinate levels are linked to higher cardiovascular disease risk factors in young adults

Background: Succinate is produced by both host and microbiota, with a key role in the interplay of immunity and metabolism and an emerging role as a biomarker for infammatory and metabolic disorders in middle-aged adults. The relationship between plasma succinate levels and cardiovascular disease (CVD) risk in young adults is unknown. Methods: Cross-sectional study in 100 (65% women) individuals aged 18–25 years from the ACTIvating Brown Adipose Tissue through Exercise (ACTIBATE) study cohort. CVD risk factors, body composition, dietary intake, basal metabolic rate, and cardiorespiratory ftness were assessed by routine methods. Plasma succinate was measured with an enzyme-based assay. Brown adipose tissue (BAT) was evaluated by positron emission tomography, and circulating oxylipins were assessed by targeted metabolomics. Fecal microbiota composition was analyzed in a sub-sample. Results: Individuals with higher succinate levels had higher levels of visceral adipose tissue (VAT) mass (+42.5%), tri‑ glycerides (+63.9%), C-reactive protein (+124.2%), diastolic blood pressure (+5.5%), and pro-infammatory omega-6 oxylipins than individuals with lower succinate levels. Succinate levels were also higher in metabolically unhealthy individuals than in healthy overweight/obese peers. Succinate levels were not associated with BAT volume or activity or with fecal microbiota composition and diversity. Conclusions: Plasma succinate levels are linked to a specifc pro-infammatory omega-6 signature pattern and higher VAT levels, and seem to refect the cardiovascular status of young adults.Spanish Ministry of Economy and Competitiveness via Retos de la Sociedad (DEP2016-79512-R to JRR and RTI2018-093919-B to SFV)European Regional Development Funds (ERDF)Spanish Ministry of Education (FPU16/02828, FPU16/05159, FPU17/01523 and FPU19/01609)University of Granada Plan Propio de Investigación 2016-Excellence actions–Unit of Excellence on Exercise and Health (UCEES)Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades (ERDF: ref. SOMM17/6107/UGR)The Spanish Ministry of Science and Innovation (PI20/00095 to VCM and PI20/00338 to JV) co-fnanced by the European Regional Development Fund (ERDF)Ramón y Cajal program (RYC2019026490-I) from the Spanish Ministry of Science and Innovation, co-fnanced by the ERDFy Fundación Bancaria Caixa d’Estalvis i Pensions de Barcelona (HR20-00051 to S.F.-V.)The Netherlands CardioVascular Research Initiative: ‘the Dutch Heart Foundation, Dutch Federation of University Medical Centers, The Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences’ (CVON2017-20 GENIUS-2) to PCNRChinese Scholarship Council (CSC, No. 201707060012) to XDMiguel Servet tenure-track program (CP10/00438 and CPII16/00008) from the Fondo de Investigación Sanitaria, co-fnanced by the ERDFFundación Alfonso Martin Escuder

Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis

PURPOSE: To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors. EXPERIMENTAL DESIGN: Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n = 49), without LC (n = 26), and undetermined (n = 3). RESULTS: FCI described a wide range of EpCAM-positive cells with a hyperdiploid DNA content in the CSF of patients with LC. Compared with cytology, FCI showed higher sensitivity (75.5 vs 65.3) and negative predictive value (67.6 vs 60.5), and similar specificity (96.1 vs 100) and positive predictive value (97.4 vs 100). Concordance between cytology and FCI was high (Kp = 0.83), although misdiagnosis of LC did not show differences between evaluating the CSF with 1 or 2 techniques (P = .06). Receiver-operator characteristic curve analyses showed that lymphocytes and monocytes had a different distribution between patients with and without LC. CONCLUSION: FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis

Minimal state models for ionic channels involved in glucagon secretion

Pancreatic alpha cells synthesize and release glucagon. This hormone along with insulin, preserves blood glucose levels within a physiological range. During low glucose levels, alpha cells exhibit electrical activity related to glucagon secretion. In this paper, we introduce minimal state models for those ionic channels involved in this electrical activity in mice alpha cells. For estimation of model parameters, we use Monte Carlo algorithms to fit steadystate channel currents. Then, we simulate dynamic ionic currents following experimental protocols. Our aims are 1) To understand the individual ionic channel functioning and modulation that could affect glucagon secretion, and 2) To simulate ionic currents actually measured in voltage-clamp alpha-cell experiments in mice. Our estimations indicate that alpha cells are highly permeable to sodium and potassium which mainly manage action potentials. We have also found that our estimated N-type calcium channel population and density in alpha cells is in good agreement to those reported for L-type calcium channels in beta cells. This finding is strongly relevant since both, L-type and N-type calcium channels, play a main role in insulin and glucagon secretion, respectively

Identification of TNF-α and MMP-9 as potential baseline predictive serum markers of sunitinib activity in patients with renal cell carcinoma using a human cytokine array

BACKGROUND: Several drugs are available to treat metastatic renal-cell carcinoma (MRCC), and predictive markers to identify the most adequate treatment for each patient are needed. Our objective was to identify potential predictive markers of sunitinib activity in MRCC. METHODS: We collected sequential serum samples from 31 patients treated with sunitinib. Sera of six patients with extreme phenotypes of either marked responses or clear progressions were analysed with a Human Cytokine Array which evaluates 174 cytokines before and after treatment. Variations in cytokine signal intensity were compared between both groups and the most relevant cytokines were assessed by ELISA in all the patients. RESULTS: Twenty-seven of the 174 cytokines varied significantly between both groups. Five of them (TNF-alpha, MMP-9, ICAM-1, BDNF and SDF-1) were assessed by ELISA in 21 evaluable patients. TNF-alpha and MMP-9 baseline levels were significantly increased in non-responders and significantly associated with reduced overall survival and time-to-progression, respectively. The area under the ROC curves for TNF-alpha and MMP-9 as predictive markers of sunitinib activity were 0.83 and 0.77. CONCLUSION: Baseline levels of TNF-alpha and MMP-9 warrant further study as predictive markers of sunitinib activity in MRCC. Selection of patients with extreme phenotypes seems a valid method to identify potential predictive factors of response

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease