Confined Cell Migration and Asymmetric Hydraulic Environments to Evaluate the Metastatic Potential of Cancer Cells.

Metastasis, a hallmark of cancer development, is also the leading reason for most cancer-related deaths. Furthermore, cancer cells are highly adaptable to micro-environments and can migrate along pre-existing channel-like tracks of anatomical structures. However, more representative three-dimensional models are required to reproduce the heterogeneity of metastatic cell migration in vivo to further understand the metastasis mechanism and develop novel therapeutic strategies against it. Here, we designed and fabricated different microfluidic-based devices that recreate confined migration and diverse environments with asymmetric hydraulic resistances. Our results show different migratory potential between metastatic and nonmetastatic cancer cells in confined environments. Moreover, although nonmetastatic cells have not been tested against barotaxis due to their low migration capacity, metastatic cells present an enhanced preference to migrate through the lowest resistance path, being sensitive to barotaxis. This device, approaching the study of metastasis capability based on confined cell migration and barotactic cell decisions, may pave the way for the implementation of such technology to determine and screen the metastatic potential of certain cancer cells.

Spice and Herb Frauds: Types, Incidence, and Detection: The State of the Art.

There is a necessity to protect the quality and authenticity of herbs and spices because of the increase in the fraud and adulteration incidence during the last 30 years. There are several aspects that make herbs and spices quite vulnerable to fraud and adulteration, including their positive and desirable sensorial and health-related properties, the form in which they are sold, which is mostly powdered, and their economic relevance around the world, even in developing countries. For these reasons, sensitive, rapid, and reliable techniques are needed to verify the authenticity of these agri-food products and implement effective adulteration prevention measures. This review highlights why spices and herbs are highly valued ingredients, their economic importance, and the official quality schemes to protect their quality and authenticity. In addition to this, the type of frauds that can take place with spices and herbs have been disclosed, and the fraud incidence and an overview of scientific articles related to fraud and adulteration based on the Rapid Alert System Feed and Food (RASFF) and the Web of Science databases, respectively, during the last 30 years, is carried out here. Next, the methods used to detect adulterants in spices and herbs are reviewed, with DNA-based techniques and mainly spectroscopy and image analysis methods being the most recommended. Finally, the available adulteration prevention measurements for spices and herbs are presented, and future perspectives are also discussed.

Borrelia burgdorferi modulates the physical forces and immunity signaling in endothelial cells.

Borrelia burgdorferi (Bb), a vector-borne bacterial pathogen and the causative agent of Lyme disease, can spread to distant tissues in the human host by traveling in and through monolayers of endothelial cells (ECs) lining the vasculature. To examine whether Bb alters the physical forces of ECs to promote its dissemination, we exposed ECs to Bb and observed a sharp and transient increase in EC traction and intercellular forces, followed by a prolonged decrease in EC motility and physical forces. All variables returned to baseline at 24 h after exposure. RNA sequencing analysis revealed an upregulation of innate immune signaling pathways during early but not late Bb exposure. Exposure of ECs to heat-inactivated Bb recapitulated only the early weakening of EC mechanotransduction. The differential responses to live versus heat-inactivated Bb indicate a tight interplay between innate immune signaling and physical forces in host ECs and suggest their active modulation by Bb.

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells.

Cell competition refers to the mechanism whereby less fit cells ("losers") are sensed and eliminated by more fit neighboring cells ("winners") and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected "loser" cells by uninfected "winner" cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.

Role of an autochthonous starter culture and the protease EPg222 on the sensory and safety properties of a traditional Iberian dry-fermented sausage "salchichón".

The effect of the addition of an autochthonous starter culture and the protease EPg222 on the physico-chemical and sensory characteristics of dry-fermented sausage ''salchichon" was investigated. Sausages were prepared with purified EPg222 and Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34 as starter culture (P200S34), separately and together, ripened for 90 days, and compared with a control batch. Dry-fermented sausages ripened with EPg222 and starter culture showed higher amounts of AN and volatile compounds derived from amino acid catabolism than the control, especially in samples in which was added the association of enzyme and starter culture (P200S34+EPg222). There were clear differences shown by the texture analysis, with the P200S34+EPg222 batch being less hard. Especially important was the result found in biogenic amines, since the association P200S34+EPg222 reduced their accumulation compared to the EPg222 batch. The use of EPg222 may be of great interest to improve the sensory characteristics of dry-fermented sausages, but its association with the selected starter culture with low decarboxylase activity is necessary to guarantee healthiness and homogeneity.

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially infected epithelial cells.

Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.

Two-year follow-up of a prospective study of circulating regulatory T cells in renal transplant patients.

CD4(+)CD25(high)FOXP3(+) regulatory T cells (Tregs) are involved in alloreactivity and may be associated with protection from rejection. Their quantification in peripheral blood could guide clinicians in the management of renal transplant patients. Thus, we prospectively monitored the levels and in vitro suppression of circulating Tregs in 33 renal transplant patients from deceased donors within the first two yr of transplantation. Patients received maintenance immunosuppression with tacrolimus, mofetil mycophenolate and prednisolone. Results showed that peripheral blood Tregs were significantly lower six months after transplantation and recovered to almost basal levels at first post-transplant year. The number of circulating Tregs increased significantly over basal levels afterwards. The decrease in circulating Tregs at six months may be explained by the high load of tacrolimus, as demonstrated by the inverse correlation between the blood concentration of Tregs and tacrolimus. Likewise, nine patients treated with anti-CD25 antibodies showed higher numbers of Tregs at six months than those that did not, although differences were not observed later. In conclusion, circulating Tregs decrease in the first six months but recover thereafter up to two yr after kidney transplantation. Such a decrease is favored by high levels of tacrolimus but not by induction protocols with anti-CD25.

Characterization of molds isolated from smoked paprika by PCR-RFLP and micellar electrokinetic capillary electrophoresis.

Molds are common contaminants of paprika meat products. The drying and storage stages of paprika processing are critical because they can provide molds with the conditions particularly appropriate for their growth and proliferation. Thus, an efficient and accurate characterization of the toxigenic molds of paprika is necessary. An RFLP analysis of the rRNA genes was performed by using a TaqI restriction enzyme. In addition, a micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by mold strains commonly found in paprika. This study was confirmed with a 5.8S-ITS region sequence analysis. A total of 31 isolates were identified by RFLP and MECC analysis. These showed stable RFLP profiles that were clearly different for the different genera and species, and were grouped into clusters together with the profiles of the 16 reference strains. MECC analysis provided additional characteristic peak patterns for the characterization of the mold species present. The characterized isolates were species of the genera Fusarium spp., Aspergillus spp., Penicillium spp., Cladosporium spp., Mucor spp. and Phlebia spp. The identifications were confirmed by the 5.8S-ITS region sequence analysis and by a BLAST search of the GenBank database. RFLP patterns with TaqI restriction enzyme and MECC profiles, either singly or combined, could be of great interest to distinguish molds in paprika.

Differentiation of Staphylococci from Iberian dry fermented sausages by protein fingerprinting.

The Staphylococci populations in different types of Iberian dry fermented sausages from central-west Spain were identified. A simple electrophoretic method of whole-cell proteins and extracellular protein profiling was evaluated for speed of identification. This study was correlated with a 16S rRNA gene sequence analysis and biochemical identification by API Staph. A total of 81 isolates were identified by SDS-PAGE of the whole-cell proteins. These showed stable profiles in the range 99-14kDa that were clearly different for the different species, and were grouped into clusters together with the profiles of the eight reference strains. SDS-PAGE of the extracellular protein extracts provided additional characteristic banding patterns for the characterization of the Staphylococcus species present. The whole-cell SDS-PAGE showed that the predominant species was Staphylococcus saprophyticus (61.7%) followed by Staphylococcus aureus (19.7%). The identifications were confirmed by the 16S rRNA gene sequencing and by a BLAST search of the GenBank database. However, the API Staph biochemical identifications were frequently erroneous at the species level. In sum, SDS-PAGE analysis showed itself to be rapid and accurate in identifying the most commonly encountered Staphylococcus isolates in dry fermented sausages.

Authentication of "Cereza del Jerte" sweet cherry varieties by free zone capillary electrophoresis (FZCE).

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used as an alternative to other methods in the determination of sweet cherry varieties for the authentication of "Cereza del Jerte". Two autochthonous varieties of sweet cherry type "Picota", 'Ambrunés' and 'Pico Negro', and the foreign variety 'Sweetheart' were used in the study. Two protocols for extracting the methanol-soluble proteins were tested. On the basis of the results, direct evaporation with nitrogen of a methanol extract was included in the extraction protocol for routine analysis. This method was found to give excellent repeatability of the corrected migration time (CMT), and showed greater effectiveness in discriminating sweet cherry varieties than the SDS-PAGE technique. Three peaks found in the FZCE electropherograms were investigated as a basis for discriminating between varieties. In addition, the FZCE analysis of methanol-soluble proteins provides information about the physico-chemical parameters relevant to the sensorial quality of the sweet cherries.

Characterisation of microbial deep spoilage in Iberian dry-cured ham.

The purpose of this work was to investigate the micro-organisms involved in overlooked "bone taint" spoilage of dry-cured Iberian hams. The physico-chemical characteristics of spoiled hams with 12 and 24 months of ripening, showing initial signs of alteration, were analyzed and their correlations with microbial counts studied. The spoilage potential of different microbial groups was assessed by the relationship between the microbial counts and the proteolysis level of spoilage as observed in the degradation of myofribrillar and sarcoplasmic protein fractions and in the changes in free amino acids. Non-enteric gram-negative bacteria (NEGN) were the dominant microbial group, showing a positive correlation with the moisture of spoiled hams. The Catalase-positive cocci (GPCP) growth was favoured by high NaCl concentrations in the spoiled hams, whereas the counts of Enterobacteriaceae were negatively affected by high NaCl concentration. The highest proteolytic microorganisms were the Gram-negative microbial groups playing Enterobacteriaceae a major role in the undesirable changes of the texture properties of the spoiled hams. With respect to the sensorial analysis, a synergy between NEGN and GPCP was observed in most of the strongly spoiled samples.

Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages.

The populations of lactic acid bacteria (LAB) in different types of Iberian dry-fermented sausages from central-west Spain were identified. A simple and rapid electrophoretic method of whole-cell protein profiles was evaluated, correlating it with 16S rRNA gene sequence analysis and biochemical identification by API 50 CHL. A total of 96 isolates were identified by SDS-PAGE showing stable profiles corresponding to 30-45 polypeptides in the range 95-8kDa that were clearly different for the different species and were grouped with those of the 9 reference strains used in this study. The SDS-PAGE method showed that the predominant species were Pediococcus acidilactici (48%) followed by Lactobacillus plantarum (23%) and Lactobacillus brevis (18%). The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. However, biochemical identifications by API 50 CHL showed different errors at the genus and species level. In sum, the SDS-PAGE analysis showed itself to be a rapid and accurate differentiation method for the most commonly encountered LAB isolates in dry-fermented sausages.

Integration of in vitro and in silico Models Using Bayesian Optimization With an Application to Stochastic Modeling of Mesenchymal 3D Cell Migration.

Cellular migration plays a crucial role in many aspects of life and development. In this paper, we propose a computational model of 3D migration that is solved by means of the tau-leaping algorithm and whose parameters have been calibrated using Bayesian optimization. Our main focus is two-fold: to optimize the numerical performance of the mechano-chemical model as well as to automate the calibration process of in silico models using Bayesian optimization. The presented mechano-chemical model allows us to simulate the stochastic behavior of our chemically reacting system in combination with mechanical constraints due to the surrounding collagen-based matrix. This numerical model has been used to simulate fibroblast migration. Moreover, we have performed in vitro analysis of migrating fibroblasts embedded in 3D collagen-based fibrous matrices (2 mg/ml). These in vitro experiments have been performed with the main objective of calibrating our model. Nine model parameters have been calibrated testing 300 different parametrizations using a completely automatic approach. Two competing evaluation metrics based on the Bhattacharyya coefficient have been defined in order to fit the model parameters. These metrics evaluate how accurately the in silico model is replicating in vitro measurements regarding the two main variables quantified in the experimental data (number of protrusions and the length of the longest protrusion). The selection of an optimal parametrization is based on the balance between the defined evaluation metrics. Results show how the calibrated model is able to predict the main features observed in the in vitro experiments.

Characterization of Micrococcaceae isolated from Iberian dry-cured sausages.

The populations of Micrococcaceae in different types of Iberian dry-cured sausages from central-west Spain were characterized and their technological and antimicrobial properties determined in order to evaluate their suitability as starter cultures in dry-cured sausage manufacture. Of a total of four hundred strains isolated from two manufacturers, one hundred and sixty-six were selected to evaluate nitrate reductase, proteolytic, lipolytic, and antimicrobial activities, and growth at different values of pH and water activity (a(w)). Most of the strains were identified as Staphylococcus except for eight isolates assigned to Kocuria spp. The species most often isolated was Staphylococccus xylosus. Others were, in descending order of abundance, S. aureus, S. lugdunensis, S. saprophyticus, S. sciuri, S. chromogenes, and S. capitis. The distributions of the minority Staphylococcus species were different for the two manufacturers. All the investigated strains were able to grow at pH and a(w) greater than 5.0 and 0.85, respectively, the values usually found in Iberian dry-cured sausages. Five S. xylosus strains showed antimicrobial activity against some indicator strains which were investigated. Seven strains with the best properties were pre-selected and tested for their lipolytic and proteolytic activities against pork fat and myofibrillar and sarcoplasmic pork proteins, respectively, and for their low biogenic amines production. Most of the strains showed proteolytic and lipolytic activities, but none produced histamine, tyramine, phenylethylamine, or spermine. Three strains, identified as Staphylococcus xylosus, possess useful properties which make them candidates for testing as starter cultures in pilot processing of Iberian sausages.

Influence of starter culture and a protease on the generation of ACE-inhibitory and antioxidant bioactive nitrogen compounds in Iberian dry-fermented sausage "salchichón".

The effect of the addition of an autochthonous starter culture and the protease EPg222 on the generation of angiotensin-I-converting enzyme (ACE)-inhibitory and antioxidant compounds by the dry-fermented sausage "salchichón" was investigated. Sausages were prepared with purified EPg222 and Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34 as the starter culture (P200S34), separately and together, ripened for 90 days, and compared to a control batch. Among the ripening time points (20, 35, 65, 90 days) studied, batches inoculated with EPg222 had higher nitrogen compound concentrations at 63 days of ripening. ACE-inhibitory and antioxidant activities were also highest in both batches with EPg222 at 63 days of ripening, and these activities were stable in most cases after in vitro simulated gastrointestinal digestion. These activities were correlated with the most relevant compounds detected by HLPC-ESI-MS. The principal components analysis (PCA) linked the P200S34 + EPg222 batch with the major compounds identified. The antioxidant activity was higher at 63 days of ripening, especially in highly proteolytic batches, such as P200S34 + EPg222. The ACE-inhibitory activity was not associated with any of the major compounds. The use of the enzyme EPg222 in association with the starter culture P200S34 in the preparation of dry-cured meat products could be of great importance due to their demonstrated ability to produce compounds with high biological activity, such as ACE-inhibitory and antioxidant activity.

Identification of fungal contamination and determination of mycotoxigenic molds by micellar electrokinetic capillary chromatography in smoked paprika.

The purpose of this work was to analyze the fungal contamination in smoked and unsmoked paprika processed from different cultivars of pepper and to investigate the ability of these and other mycotoxigenic molds to grow and synthesize mycotoxins in smoked paprika. Eighteen mycotoxins were evaluated using micellar electrokinetic capillary chromatography. No relevant differences were found in fungal contamination between smoked and unsmoked paprika. The number of yeasts obtained was low, ranging from 0.4 to 3.29 log CFU g(-1); most of the yeast strains were identified as Cryptococcus spp. followed by Candida spp. All mold counts were <4 log CFU g(-1). Aspergillus, Cladosporium, Penicillium, and Fusarium were the predominant hyphomycete genera. Six mycotoxins were identified in the extracts of several strains isolated from paprika and incubated on malt extract agar. Penicillium expansum followed by Penicillium citrinum and Penicillium raistrickii were the dominant mycotoxigenic fungi isolated. Most of themycotoxin-producing fungi produced detectable amounts of mycotoxins when grown on paprika agar.

Anti-ribosomal P protein autoantibodies from patients with neuropsychiatric lupus impair memory in mice.

OBJECTIVE: To define whether anti-ribosomal P (anti-P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood. METHODS: We used anti-P antibodies from patients with NPSLE and rabbit-generated anti-P and anti-NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal-dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti-P or anti-NSPA, using lipopolysaccharide (LPS) to permeate the blood-brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays. RESULTS: Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti-P and anti-NSPA staining colocalization, and responded to both anti-P and anti-NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti-P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti-P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti-NSPA antibodies also impaired memory. CONCLUSION: Anti-P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti-P can access the hippocampus and impair memory without requiring neuronal death when the blood-brain barrier is disrupted. NSPA can mediate antibody-driven diffuse brain dysfunction, and anti-P might contribute to the cognitive impairment that is frequently observed in SLE.

Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins.

Moulds grow on many different dry-cured meat products and are able to hydrolyse muscle proteins. However, their contribution to proteolysis in these products is not well known. Only recently, the ability of just a few strains of Penicillium spp. to increase proteolysis in dry-cured meat products has been shown. For these strains to be used as starter cultures, their hydrolytic activity under standard conditions should be characterised. With this purpose, the effect of Penicillium chrysogenum Pg222 on pork myofibrillar proteins has been assayed in a culture medium containing 5% (w/v) NaCl. SDS-PAGE revealed that Pg222 was responsible for extensive hydrolysis of the main myofibrillar proteins except alpha-actinin. The proteolysis led to increases in free amino acids, reaching peak values at 84 h. Ala, Tyr and Lys were present in the greatest amount. These results suggest that P. chrysogenum Pg222 would contribute to development of desired texture and flavours in dry-cured meat products.

Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins.

During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development. However, their contribution to volatile components of dry-cured meat products is not known. One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening. Sterile pork loins were inoculated and ripened for 106 days. Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS. Inoculation of pork loins with P. chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids. Inoculation with D. hansenii seemed to favour the formation of complex alcohols.

Contribution of a selected fungal population to proteolysis on dry-cured ham.

The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids. Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds. Several months of ripening time are required to allow the particular flavour to develop. The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages. The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham. For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures. Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams. Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing. However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams. In addition, the concentration for some of the more polar free amino acids (i.e. Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles. These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham.