Pure Cell Cultures Research Articles

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation — a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Onion (Allium cepa L.) Skin Waste Valorization: Unveiling the Phenolic Profile and Biological Potential for the Creation of Bioactive Agents through Subcritical Water Extraction.

Onion skin waste (OSW), the primary non-edible byproduct from onion processing, offers a renewable source of bioactive compounds. This study aims to valorize OSW through subcritical water extraction (SWE), aligning with a circular economy and biorefinery principles. SWE was carried out at 145 °C and 50 bar for 50 min in a discontinuous reactor, producing a phenolic-rich extract (32.3 ± 2.6 mg/g) dominated by protocatechuic acid (20.3 ± 2.5 mg/g), quercetin-4'-O-glucoside (7.5 ± 0.2 mg/g), and quercetin (3.2 ± 0.6 mg/g). Additionally, the extract contains sugars (207.1 ± 20.3 mg sucrose-Eq/g), proteins (22.8 ± 1.6 mg BSA-Eq/g), and free amino acids (20.4 ± 1.2 mg arginine-Eq/g). Its phenolic richness determines its scavenging activity against ●NO and O2●- radicals and its α-glucosidase and aldose-reductase inhibition without affecting α-amylase. Notably, the extract demonstrates significant α-glucosidase inhibition (IC50 = 75.6 ± 43.5 µg/mL), surpassing acarbose (IC50 = 129.5 ± 1.0 µg/mL) in both pure enzyme and cell culture tests without showing cytotoxicity to AGS, HepG2, and Caco-2 human cell lines. The extract's bioactivity and nutritional content make it suitable for developing antioxidant and antidiabetic nutraceutical/food components, highlighting SWE's potential for OSW valorization without using organic solvents.

P009 IL-17 mediates visceral hypersensitivity by sensitising colonic nociceptors

Abstract Background Comparison of cytokine expression in colitic tissue with respective cytokine receptor expression in pain sensing neurons highlights a likely contribution of IL-17 to the production of abdominal pain during flare. To explore this pro-nociceptive potential of IL-17 in colitis we examined the ability of IL-17 to stimulate sensory neurons and the colonic afferent response to noxious distension. Methods IL-17 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to IL-17 (10-300 ng/mL) alone and following pre-treatment with either TRPV1 or TRPA1 inhibitors. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. In a separate set of experiments the effect of IL-17 was also examine on a neuron pure cell culture in which non-neuronal cells were removes by magnet assisted cell sorting. IL-17 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and the response to noxious luminal distension of the isolated colorectum (ex-vivo), perfused with carbogenated Krebs solution, examined following pre-treatment with IL-17 (50 ng/mL) alone or in the presence of respective inhibitors. All experiments were performed using tissue from male C57BL/6J mice euthanaised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Consistent with the ability of IL-17 to stimulate sensory neurons including those classified as nociceptors by their co-sensitivity to capsaicin, pre-treatment with IL-17 also produced a significant sensitisation of the colonic afferent response to luminal distension at noxious distending pressures. The increase in [Ca2+]i produced by IL-17 in sensory neurons was blocked by pre-treament with a TRPV1 antagonist but not a TRPA1 antagonist. Furthermore, the proportion of neurons stimulated by IL-17 was comparable between standard DRG cell cultures and neuron pure cell cultures demonstrating a direct effect of IL-17 on sensory neurons. Finally, IL-17 mediated mechanosensitisation of colonic nociceptors was unaffected by TRPV1 inhibition. Conclusion Our findings demonstrate that IL-17 stimulates capsaicin-sensitive nociceptors and mediates visceral hypersensitivity in colonic nociceptors consistent with a causative role for IL-17 is the generation of abdominal pain in people with colitis.

A comparative analysis of genes differentially expressed between rete testis cells and Sertoli cells of the mouse testis

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Peracetic acid mode-of-action on aquaculture microbes evaluated by dual-staining flow cytometry

Flow cytometry with dual staining allows an exact quantification of live and dead cells. It is a potential tool for gaining new information on the mode-of-action of disinfection chemicals and supporting efficient microbial control in recirculating aquaculture systems (RAS). Here, we used a dual-staining flow cytometry to examine the disinfection efficiency of peracetic acid (PAA) against i) the fish pathogenic bacterium Aeromonas salmonicida and ii) on microbes present in RAS. The method allowed us to accurately quantify the A. salmonicida cell response to different PAA concentrations (1, 2, and 10 mg/L) in time and to calculate the PAA concentration needed to achieve 95% cell mortality (LC95 estimates). With the high PAA dose of 10 mg/L, LC95 was reached in <15 min, while with the intermediate dose of 2 mg/L, LC95 was reached only in the end of the 60 min trial and with the low 1 mg/L dose, the disinfection efficiency remained low. The addition of soluble organic matter increased the PAA decay and decreased the disinfection efficiency. In RAS water, disinfection efficiency increased with increasing PAA concentration (from 1 to 10 mg/L) but was significantly reduced with increasing organic matter concentration (TCOD from 7.6 to 30.3 mg O2/L). The increase in the proportion of dead cells was found to be a reliable proxy for disinfection efficiency in RAS water. In the pure cell culture, both the increase in the proportion of dead cells and the fraction of reduced live cells reflected the disinfection efficiency of PAA. Altogether, the study demonstrated the PAA mode-of-action on aquaculture microbes and showed that flow cytometry with dual staining allows fast and precise examination of microbial viability under different conditions.

The Impact of Microelectrode Pattern on the Sensitivity of Tracing Environmental CO2 Deficiency in Cellular Metabolism by a New Design of Electrochemical Biosensor.

Here, two different electrode patterns are described as cyclic voltammetry (CV) biosensors to detect the effect of a hypo CO2 condition (for 6 h) in ambient on cellular secretion. The cells were selected from breast cancer and endothelial standard lines. Changes in CV peaks of the secretions were recorded by the modified pattern whereby increasing the interactive surface with homogenous electric paths was considered by simulation before fabrication. The results of the simulation and experimental procedures showed a meaningful correlation between hypo CO2 samples and the occurrence of CV oxidative peaks at about 0.07 V and reductive peaks at approximately -0.22 V in the modified biosensor in all cell lines, while no apoptosis was found in any of the control and hypo CO2 samples. This observation could not be related to the lack of H+ (alkaline pH induction) in the media solution as such peaks were not observed in the pure cell culture medium but had been maintained in the hypo CO2 ambient. This approach could be used as a cell-free sensor to monitor ambient shocks. This may not induce apoptosis but may be vital in the proliferation and protein expression of the cells, such as the hypo CO2 ambient. The sensor is not disposable in use and showed repeatable responses after rinsing.

Assessing the Efficacy, Acute Toxicity, and Binding Modes of the Agricultural Nitrification Inhibitors 3,4-Dimethyl-1H-pyrazole (DMP) and Dicyandiamide (DCD) with Nitrosomonas europaea

Nitrification inhibitors have been coformulated with nitrogen fertilizers since the 1970s to modulate the microbiological conversion of nitrogen in agricultural soils. 3,4-Dimethyl-1H-pyrazole (DMP) and dicyandiamide (DCD) are currently the most used commercial nitrification inhibitors, but their mode of action is not well understood. This work seeks to fill this void by assessing for the first time in detail their mechanism of inhibition, efficacy, and acute toxicity with pure cell cultures of Nitrosomonas europaea. Bacterial assays based on the quantification of the nitrite (NO2–) production showed that both inhibitors reversibly target ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Michaelis–Menten kinetics suggest that both DMP and DCD act as uncompetitive inhibitors. Real-time measurements of the oxygen (O2) consumption confirmed the nonmechanistic mode of inhibition and showed that DMP reduced the O2 uptake rate by AMO much more at considerably lower concentrations than DCD, in line with the lower inhibitory efficiency of the latter. Acute toxicity tests revealed that DCD has a 10% higher toxicity than DMP when comparing treatments at the same inhibition efficacy (i.e., DMP at 10 ppm, DCD at 100 ppm), indicating that the inhibition of the nitrification process cannot simply be achieved by increasing the inhibitor concentration. The methods presented in this study could assist the development of more reliable nitrification inhibitors in the future.

Role of geneticin in isolation and culturing of skin melanocytes and melanoma cells

Abstract Introduction Development of an effective, suitable, and reliable method for both the isolation and culturing of melanocytes is crucial for studies on pathomechanisms of skin diseases originating from melanocyte disorders. In this study, we have investigated the influence of geneticin (G418), a substance used for melanocyte selection, in the view of the frequency of presence of cells such as keratinocytes and fibroblasts, widely known as contaminators of melanocyte-originating cell cultures. Materials and Methods Study was conducted on primary, freshly isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells as well as on commercially available melanoma cell lines MeWo, G-361, and A375. Cells were cultured in different culture media supplemented with various concentrations of geneticin ranging from 0.05 to 1 mg/mL. Cell viability, proliferation rate and detection of apoptotic/necrotic cells was assessed. Results Choice of culture media supplemented with various concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) strongly affect viability of melanocytes, fibroblasts, and keratinocytes. Selective culture media without FBS facilitate the process of melanocytes and melanoma pure cell culture, yet without geneticin supplementation are insufficient for complete eradication of fibroblast contamination from cell culture. Conclusions In this study we provide, for the first time, the dose-response action of keratinocytes and fibroblasts upon geneticin stimulation in different culture media and show that a low concentration (0.05 mg/mL) of geneticin added to the selective culture media may be safely implemented to facilitate the production of melanocyte and melanoma cell cultures that are free from frequent cell contaminants.

Features of the &lt;i&gt;Candida&lt;/i&gt; genus community pattern in the intestinal biotope of patients with tuberculosis

Fungi are opportunistic microorganisms that colonize all biotopes of the human body, including intestinal. In case of emerging adverse environmental factors (HIV infection, other immunodeficiencies, antibiotic therapy), these microbial representatives begin active reproduction, which might require prescribing antimycotics. Frequent use of the latter in clinical practice induces the development of drug resistance to antifungal drugs, which may impact on effectiveness of both the treatment of fungal infections and other diseases. The purpose of the study was to assess the pattern and spectrum of drug resistance of Candida genus in the intestinal biotope of patients with respiratory tuberculosis and identify risk factors for developing total fungal drug resistance to antimycotic drugs. Material and methods. There were enrolled 21 patients with respiratory tuberculosis. Pattern of the fungal species diversity for the Candida genus isolated from faeces was evaluated, and the spectrum of relevant drug resistance to antimycotic drugs was determined. Patients (n = 21) were divided into 2 groups: with (n = 10) and without (n = 11) total resistance to antimycotics, after which the main risk factors for its development were identified. Results. Members of the Candida genus were isolated from all patients examined, wherein pure cell cultures were characterized by high level of antimycotics resistance. Resistance to three drugs was noted in 1 culture (4.8%), to four in 10 cultures (47.6%), also found in 10 cultures to the entire drug panel (47.6%). During statistical processing, the data were obtained on affecting formation of total resistance to antimycotics of concomitant pathology of the gastrointestinal tract, the presence of a clinically significant dyspeptic syndrome, a history of antimycotic therapy, HIV infection with severe immunodeficiency, and some decrease in the peripheral blood CD4+ lymphocyte count. Conclusion. Fungi of the Candida genus isolated from tuberculosis patients were characterized by high level of resistance to antimycotics. Total resistance was observed in 47.6% of patients. In addition, the major fungi colonizing the intestines of tuberculosis patients were found to be Candida albicans species. The risk factors for the development of total antifungal resistance included: chronic enterocolitis, dyspeptic syndrome, peripheral blood CD4+ lymphocyte count lower than 350 cells/l, and history of antimycotic therapy.

Spectrophotometry and colorimetry profiling of pure phenol red and cell culture medium on pH variation

AbstractThe application of phenol red has been widely applied in cell culture media to enable monitoring of the pH value of cell growth. However, sometimes it is difficult for a human observer to identify the colour transformation using only the naked eye. Furthermore, different people will give different interpretations of colour transformation. Thus the current study was performed to provide the spectrophotometry and colorimetry profiling of pure phenol red and cell culture media to pH variation. Pure phenol red and cell culture media were used as samples. The pH values of both types of sample were manipulated using citric acid. Spectrophotometry analysis results show that the pH value of the pure phenol red samples could be well profiled by using the wavelengths in the green light region (in the range of 500‐600 nm). The pH value of the cell culture medium samples could also be well profiled, by using wavelengths in either the green or blue light region. Colorimetry analysis results conducted using CIELab colour space indicate that the colour transformation of both samples is linear, with a high value for the coefficient of determination. This profiling proves that phenol red can be used to quantify the pH values of the samples and shows good potential to be applied in the development of spectroscopy‐based and colorimetry‐based pH sensors.

Establishment of a pure culture of immature Sertoli cells by PDGFRA staining and cell sorting.

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies.

Effects of the Probiotic Activity of Bacillus subtilis DSM 29784 in Cultures and Feeding Stuff.

The European Union banned the usage of antibiotic growth promoters in animal production. The probiotic microorganism of the genus Bacillus appeared to be an attractive candidate to replace antibiotics. The Bacillus subtilis DSM 29784 is one of these strains. To date, the probiotic effect has not been completely understood, but it is supposed that the effect depends on metabolites of the microorganism. Imaging high-performance thin-layer chromatography (HPTLC) is a powerful tool to visualize differences in the metabolite profile of bacteria with high genetic similarity to allow a better understanding of the probiotic effect. In comparison to other bacteria, especially these bacterial cells were more robust to harsh cultivation conditions and produced a higher level of antioxidants or bioactive substances such as surfactin. HPTLC enabled the comparison of pure cell cultures to the spore cultivation in the feed, and the results explain and support the probiotic effect.

Role of TGF\u03b21 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Primary Chicken and Duck Endothelial Cells Display a Differential Response to Infection with Highly Pathogenic Avian Influenza Virus.

Highly pathogenic avian influenza viruses (HPAIVs) in gallinaceous poultry are associated with viral infection of the endothelium, the induction of a ‘cytokine storm, and severe disease. In contrast, in Pekin ducks, HPAIVs are rarely endothelial tropic, and a cytokine storm is not observed. To date, understanding these species-dependent differences in pathogenesis has been hampered by the absence of a pure culture of duck and chicken endothelial cells. Here, we use our recently established in vitro cultures of duck and chicken aortic endothelial cells to investigate species-dependent differences in the response of endothelial cells to HPAIV H5N1 infection. We demonstrate that chicken and duck endothelial cells display a different transcriptional response to HPAI H5N1 infection in vitro—with chickens displaying a more pro-inflammatory response to infection. As similar observations were recorded following in vitro stimulation with the viral mimetic polyI:C, these findings were not specific to an HPAIV H5N1 infection. However, similar species-dependent differences in the transcriptional response to polyI:C were not observed in avian fibroblasts. Taken together, these data demonstrate that chicken and duck endothelial cells display a different response to HPAIV H5N1 infection, and this may help account for the species-dependent differences observed in inflammation in vivo.

Calcium phosphate/polyvinyl acetate coatings on SS304 via galvanic co-deposition for orthopedic implant applications

Abstract In this work, the galvanic deposition method is used to deposit coatings of brushite/hydroxyapatite/polyvinyl acetate on 304 stainless steel. Coatings are obtained at different temperatures and with different sacrificial anodes, consisting of a mixture of brushite and hydroxyapatite. Samples are aged in a simulated body fluid (SBF), where a complete conversion of brushite into hydroxyapatite with a simultaneous change in morphology and wettability occurred. The corrosion tests show that, compared with bare 304, the coating shifts Ecorr to anodic values and reduces icorr Ecorr, and icorr has different values at different aging times due to chemical interactions at the solid/liquid interface. The best performing deposits are those obtained by using Al as the sacrificial anode. The metal ion release, measured after 21 days of aging, is very low and is attributable to the presence of a coating that slows the steel corrosion. Coating cytotoxicity is investigated through cell viability assays with MC3T3-E1 osteoblastic cells. The results reveal a high cytocompatibility comparable to that of a pure cell culture medium.

Characterization of rodent Sertoli cell primary cultures.

Sertoli cells play a vital role in spermatogenesis by offering physical and nutritional support to the differentiating male germ cells. They form the blood-testis barrier and secrete growth factors essential for germ cell differentiation. Sertoli cell primary cultures are critical for understanding the regulation of spermatogenesis; however, obtaining pure cultures has been a challenge. Rodent Sertoli cell isolation protocols do not rule out contamination by the interstitial or connective tissue cells. Sertoli cell-specific markers could be helpful, but there is no consensus. Vimentin, the most commonly used marker, is not specific for Sertoli cells since its expression has been reported in peritubular myoid cells, mesenchymal stem cells, fibroblasts, macrophages, and endothelial cells, which contaminate Sertoli cell preparations. Markers based on transcription and growth factors also have limitations. Thus, the impediment to obtaining pure Sertoli cell cultures pertains to both the method of isolation and marker usage. The aim of this review is to discuss improvements to current methods of rodent Sertoli cell primary cultures, assess the properties of prepubertal versus mature Sertoli cell cultures, and propose steps to improve cellular characterization. Potential benefits of using contemporary approaches, including lineage tracing, specific cell ablation, and RNA-seq for obtaining Sertoli-specific transcript markers are discussed. Evaluating the specificity and applicability of these markers at the protein level to characterize Sertoli cells in culture would be critical. This review is expected to positively impact future work using primary cultures of rodent Sertoli cells.

In Vitro to In Vivo Concordance of Toxicity Using the Human Proximal Tubule Cell Line HK-2.

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

Hallmarks of NLRP3 inflammasome activation are observed in organotypic hippocampal slice culture.

SummaryMicroglial inflammation driven by the NACHT, LRR and PYD domain‐containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two‐dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three‐dimensional, complex tissue architecture. Toll‐like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin‐1β expression. Nigericin‐induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase‐1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin‐1β release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development.

Vascular Wall-Mesenchymal Stem Cells Differentiation on 3D Biodegradable Highly Porous CaSi-DCPD Doped Poly (α-hydroxy) Acids Scaffolds for Bone Regeneration.

Vascularization is a crucial factor when approaching any engineered tissue. Vascular wall–mesenchymal stem cells are an excellent in vitro model to study vascular remodeling due to their strong angiogenic attitude. This study aimed to demonstrate the angiogenic potential of experimental highly porous scaffolds based on polylactic acid (PLA) or poly-e-caprolactone (PCL) doped with calcium silicates (CaSi) and dicalcium phosphate dihydrate (DCPD), namely PLA-10CaSi-10DCPD and PCL-10CaSi-10DCPD, designed for the regeneration of bone defects. Vascular wall–mesenchymal stem cells (VW-MSCs) derived from pig thoracic aorta were seeded on the scaffolds and the expression of angiogenic markers, i.e. CD90 (mesenchymal stem/stromal cell surface marker), pericyte genes α-SMA (alpha smooth muscle actin), PDGFR-β (platelet-derived growth factor receptor-β), and NG2 (neuron-glial antigen 2) was evaluated. Pure PLA and pure PCL scaffolds and cell culture plastic were used as controls (3D in vitro model vs. 2D in vitro model). The results clearly demonstrated that the vascular wall mesenchymal cells colonized the scaffolds and were metabolically active. Cells, grown in these 3D systems, showed the typical gene expression profile they have in control 2D culture, although with some main quantitative differences. DNA staining and immunofluorescence assay for alpha-tubulin confirmed a cellular presence on both scaffolds. However, VW-MSCs cultured on PLA-10CaSi-10DCPD showed an individual cells growth, whilst on PCL-10CaSi-10DCPD scaffolds VW-MSCs grew in spherical clusters. In conclusion, vascular wall mesenchymal stem cells demonstrated the ability to colonize PLA and PCL scaffolds doped with CaSi-DCPD for new vessels formation and a potential for tissue regeneration.

Microbial Protein Production from Agro-industrial Wastes as Food and Feed

Microbial protein refers to dead, dry microbial cells or total proteins extracted from pure microbial cell culture and is produced using a number of different microorganisms including bacteria, yeasts, filamentous fungi, and algae, which are used as protein supplement in human foods or animal feeds. It can also be called single cell protein, bio protein or biomass. Microorganisms, utilize inexpensive feedstock and wastes as sources of carbon and energy for growth to produce biomass, protein concentrate or amino acids. Besides high protein content (about 60-82% of dry cell weight), microbial protein also contains fats, carbohydrates, nucleic acids, vitamins and minerals like potassium, phosphorus. Another advantage with microbial protein is that it is rich in certain essential amino acids like lysine, methionine which are limiting in most plant and animal. With increase in population and worldwide protein shortage the use of microbial biomass as food and feed is more highlighted. One of the most prominent applications of microbial protein (single cell protein) is that it can be used as dietary supplements to address important issues like malnutrition especially in the developing countries.