Comparison of the Effect of Two Different Handling Conditions at Slaughter in Saliva Analytes in Pigs.

In this report, different handling conditions at slaughterhouse were studied to assess changes in salivary biomarkers. For this purpose, finishing pigs were divided into two groups, one in which handling was improved to minimize stress (Group A, n = 24, transported and stabled at the slaughterhouse at low density without mixing with unfamiliar animals throughout the whole process) and another one in which animals had a more stressful handling process (Group B, n = 24, transported and stabled at high density with unfamiliar animals). Saliva samples were taken the day before transport to the slaughterhouse at 8:00 a.m. (B0) and 12:00 a.m. (B4), and the day of slaughter just after unloading animals at the slaughterhouse at approximately 8:00 a.m. (S0) and after 4 h of lairage at approximately 12:00 a.m. (S4). Group B showed significantly higher cortisol, total esterase activity, oxytocin, adenosine deaminase and haptoglobin levels than the Group A at both S0 and S4 sampling times, and higher levels of calprotectin and creatine kinase at S4 sampling time. This report indicates that differences in the way in which the pigs are handled at the slaughterhouse can lead to changes in salivary biomarkers and opens the possibility of the use of biomarker at slaughter to monitor handling conditions.

Advances in current in vitro models on neurodegenerative diseases.

Many neurodegenerative diseases are identified but their causes and cure are far from being well-known. The problem resides in the complexity of the neural tissue and its location which hinders its easy evaluation. Although necessary in the drug discovery process, in vivo animal models need to be reduced and show relevant differences with the human tissues that guide scientists to inquire about other possible options which lead to in vitro models being explored. From organoids to organ-on-a-chips, 3D models are considered the cutting-edge technology in cell culture. Cell choice is a big parameter to take into consideration when planning an in vitro model and cells capable of mimicking both healthy and diseased tissue, such as induced pluripotent stem cells (iPSC), are recognized as good candidates. Hence, we present a critical review of the latest models used to study neurodegenerative disease, how these models have evolved introducing microfluidics platforms, 3D cell cultures, and the use of induced pluripotent cells to better mimic the neural tissue environment in pathological conditions.

Gaining knowledge about biomarkers of the immune system and inflammation in the saliva of pigs: The case of myeloperoxidase, S100A12, and ITIH4.

An assay for the measurement of myeloperoxidase (Mpx) in porcine saliva was developed and validated, and factors influencing Mpx and another two biomarkers of inflammation and immune system, the protein S100A12 and the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were studied. The spectrophotometric method for Mpx measurement validated in this assay showed an adequate analytical performance including precision and accuracy. When a group of twenty healthy pigs was sampled every 4 h from 8 a.m. until 8 p.m., Mpx and S100A12 showed significant increases at 4 p.m., whereas ITIH4 concentration showed a significant decrease at 12 a.m. Increases were also seen in salivary Mpx, S100A12, and ITIH4 levels 24 h after the intramuscular administration of Escherichia coli lipopolysaccharide in five pigs; whereas in a non-septic inflammation after the subcutaneous administration of turpentine oil to five pigs changes were seen in S100A12 at 3 h and in ITIH4 at 48 h. When a stressful situation consisting of the transportation and stay of 4 h to a slaughterhouse of 24 pigs was performed, all analytes were increased after 4 h of lairage in the slaughterhouse compared with the values that were obtained the day before at the same time of the day. Mpx can be measured in the saliva of pigs with the automated assay described in this report. Mpx, S100A12, and ITIH4 salivary levels can change depending on the hour of the day in which the sample is taken, and increases can be produced due to sepsis, non-septic inflammation and stress.

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of ß-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis.

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the latter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 µL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth).

On-chip acoustophoretic isolation of microflora including S. typhimurium from raw chicken, beef and blood samples.

Pathogen analysis in food samples routinely involves lengthy growth-based pre-enrichment and selective enrichment of food matrices to increase the ratio of pathogen to background flora. Similarly, for blood culture analysis, pathogens must be isolated and enriched from a large excess of blood cells to allow further analysis. Conventional techniques of centrifugation and filtration are cumbersome, suffer from low sample throughput, are not readily amenable to automation and carry a risk of damaging biological samples. We report on-chip acoustophoresis as a pre-analytical technique for the resolution of total microbial flora from food and blood samples. The resulting 'clarified' sample is expected to increase the performance of downstream systems for the specific detection of the pathogens. A microfluidic chip with three inlets, a central separation channel and three outlets was utilized. Samples were introduced through the side inlets, and buffer solution through the central inlet. Upon ultrasound actuation, large debris particles (10-100 µm) from meat samples were continuously partitioned into the central buffer channel, leaving the 'clarified' outer sample streams containing both, the pathogenic cells and the background flora (ca. 1 µm) to be collected over a 30 min operation cycle before further analysis. The system was successfully tested with Salmonella typhimurium-spiked (ca. 10(3)CFU mL(-1)) samples of chicken and minced beef, demonstrating a high level of the pathogen recovery (60-90%). When applied to S. typhimurium contaminated blood samples (10(7)CFU mL(-1)), acoustophoresis resulted in a high depletion (99.8%) of the red blood cells (RBC) which partitioned in the buffer stream, whilst sufficient numbers of the viable S. typhimurium remained in the outer channels for further analysis. These results indicate that the technology may provide a generic approach for pre-analytical sample preparation prior to integrated and automated downstream detection of bacterial pathogens.

On-chip processing of particles and cells via multilaminar flow streams.

The processing of particles, cells, and droplets for reactions, analyses, labeling, and coating is an important aspect of many microfluidic workflows. However, performing multi-step processes is typically a laborious and time-consuming endeavor. By exploiting the laminar nature of flow within microchannels, such procedures can benefit in terms of both speed and simplicity. This can be achieved either by manipulating the flow streams around the objects of interest, particularly for the localized perfusion of cells, or by manipulating the objects themselves within the streams via a range of forces. Here, we review the variety of methods that have been employed for performing such "multilaminar flow" procedures on particles, cells, and droplets.

Multiple flow profiles for two-phase flow in single microfluidic channels through site-selective channel coating.

An approach to control two-phase flow systems in a poly(dimethylsiloxane) (PDMS) microfluidic device using spatially selective surface modification is demonstrated. Side-by-side flows of ethanol : water solutions containing different polymers are used to selectively modify both sides of a channel by laminar flow patterning. Introduction of air pockets during modification allows for control over the length of the channel section that is modified. This approach makes it possible to achieve slug flow and side-by-side flow of water : 1-octanol simultaneously within the same PDMS channel, without the need of additional structural elements. A key finding is that conditioning of the PDMS channels with 1-octanol before polymer deposition is crucial to achieving stable side-by-side flows.