OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids.

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35-52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.

A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Electronic Nose for Improved Environmental Methane Monitoring.

Reducing emissions of the key greenhouse gas methane (CH4) is increasingly highlighted as being important to mitigate climate change. Effective emission reductions require cost-effective ways to measure CH4 to detect sources and verify that mitigation efforts work. We present here a novel approach to measure methane at atmospheric concentrations by means of a low-cost electronic nose strategy where the readings of a few sensors are combined, leading to errors down to 33 ppb and coefficients of determination, R2, up to 0.91 for in situ measurements. Data from methane, temperature, humidity, and atmospheric pressure sensors were used in customized machine learning models to account for environmental cross-effects and quantify methane in the ppm-ppb range both in indoor and outdoor conditions. The electronic nose strategy was confirmed to be versatile with improved accuracy when more reference data were supplied to the quantification model. Our results pave the way toward the use of networks of low-cost sensor systems for the monitoring of greenhouse gases.

A portable smartphone-based imaging surface plasmon resonance biosensor for allergen detection in plant-based milks.

Food allergies are hypersensitivity immune responses triggered by (traces of) allergenic compounds in foods and drinks. The recent trend towards plant-based and lactose-free diets has driven an increased consumption of plant-based milks (PBMs) with the risk of cross-contamination of various allergenic plant-based proteins during the food manufacturing process. Conventional allergen screening is usually performed in the laboratory, but portable biosensors for on-site screening of food allergens at the production site could improve quality control and food safety. Here, we developed a portable smartphone imaging surface plasmon resonance (iSPR) biosensor composed of a 3D-printed microfluidic SPR chip for the detection of total hazelnut protein (THP) in commercial PBMs and compared its instrumentation and analytical performance with a conventional benchtop SPR. The smartphone iSPR shows similar characteristic sensorgrams compared with the benchtop SPR and enables the detection of trace levels of THP in spiked PBMs with the lowest tested concentration of 0.625 µg/mL THP. The smartphone iSPR achieved LoDs of 0.53, 0.16, 0.14, 0.06, and 0.04 µg/mL THP in 10x-diluted soy, oat, rice, coconut, and almond PBMs, respectively, with good correlation with the conventional benchtop SPR system (R2 0.950-0.991). The portability and miniaturized characteristics of the smartphone iSPR biosensor platform make it promising for the future on-site detection of food allergens by food producers.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Screen-Printed Corrosion-Resistant and Long-Term Stable Stretchable Electronics Based on AgAu Microflake Conductors.

High-throughput production methods such as screen printing can bring stretchable electronics out of the lab into the market. Most stretchable conductor inks for screen printing are based on silver nanoparticles or flakes due to their favorable performance-to-cost ratio, but silver is prone to tarnishing and corrosion, thereby limiting the stability of such conductors. Here, we report on a cost-efficient and scalable approach to resolve this issue by developing screen printable inks based on silver flakes chemically coated by a thin layer of gold. The printed stretchable AgAu conductors reach a conductivity of 8500 S cm-1, remain conductive up to 250% strain, show excellent corrosion and tarnishing stability, and are used to demonstrate wearable LED and NFC circuits. The reported approach is attractive for smart clothing, as the long-term functionality of such devices is expected in a variety of environments.

Nanotechnology for Electronic Materials and Devices.

The historical scaling down of electronics devices is no longer the main goal of the International Roadmap for Devices and Systems [...].

TGFß selects for pro-stemness over pro-invasive phenotypes during cancer cell epithelial-mesenchymal transition.

Transforming growth factor ß (TGFß) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFß withdrawal and correlates with metastatic colonization. Whether TGFß promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFß induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFß withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFß receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFß suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFß treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFß-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFß to differentiate between pro-stemness and pro-invasive phenotypes.

Print-and-stick unibody microfluidics coupled surface plasmon resonance (SPR) chip for smartphone imaging SPR (Smart-iSRP).

The design of a smartphone imaging surface plasmon resonance (Smart-iSPR) system integrated with an affordable 3D-printed microfluidic SPR chip fabricated via a facile manufacturing approach could pave the way towards the development of miniaturized and integrated smartphone iSPR biosensors for emerging point-of-use applications. Conventional smartphone-based SPR systems using soft photolithography for the fabrication of microfluidics SPR chips are costly, labour-intensive and required a specially-equipped light-controlled environment, that is inadequate and mismatched with the consumer-based smartphone detection platform. Herein, we report the design, fabrication and testing of an innovative print-and-stick unibody microfluidics coupled SPR chip for smartphone iSPR. The 3D-printed microfluidics (∼€0.006) is assembled via an aptly-sized adhesive tape with the gold SPR sensing surface. Such a simple integrated microfluidic SPR chip with the print-and-stick configuration has a high resistance to fluid leakages at the channel-to-sensor interface with pressure up to 66.9 Pa and the tubing-to-inset interfaces with pressure up to 86.9 Pa. The smartphone iSPR platform weighs 138 g and with a dimension of around 70 × 60 × 40 mm3, and its performance was characterized using a standard Biacore® ß2-microglobulin calibration kit. The sensorgrams obtained by the smartphone iSPR show all the typical characteristics for surface functionalization, association and dissociation events. The smartphone iSPR responds linearly to ß2-microglobulin within the range of 10-200 nM (R2 = 0.986) with a limit-of-detection (LOD) of 1.5 nM. Given the miniaturized feature and simple camera-based imaging smartphone iSPR, the analytical performance is satisfactory when compared with the analytical dynamic range of 2-32 nM described in the Biacore® protocol.

A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface.

The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm "doublets"-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their "singlet" brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.

IgE cross-linking induces activation of human and mouse mast cell progenitors.

BACKGROUND: The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. OBJECTIVE: Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation in vivo, we hypothesized that they are activated by IgE cross-linking. METHODS: Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. CONCLUSIONS: Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

Kel1 is a phosphorylation-regulated noise suppressor of the pheromone signaling pathway.

Mechanisms have evolved that allow cells to detect signals and generate an appropriate response. The accuracy of these responses relies on the ability of cells to discriminate between signal and noise. How cells filter noise in signaling pathways is not well understood. Here, we analyze noise suppression in the yeast pheromone signaling pathway and show that the poorly characterized protein Kel1 serves as a major noise suppressor and prevents cell death. At the molecular level, Kel1 prevents spontaneous activation of the pheromone response by inhibiting membrane recruitment of Ste5 and Far1. Only a hypophosphorylated form of Kel1 suppresses signaling, reduces noise, and prevents pheromone-associated cell death, and our data indicate that the MAPK Fus3 contributes to Kel1 phosphorylation. Taken together, Kel1 serves as a phospho-regulated suppressor of the pheromone pathway to reduce noise, inhibit spontaneous activation of the pathway, regulate mating efficiency, and prevent pheromone-associated cell death.

Heimdallarchaea encodes profilin with eukaryotic-like actin regulation and polyproline binding.

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP2 and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator-stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor.

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers.

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (STm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.

Salmonella enterica Serovar Typhimurium Exploits Cycling through Epithelial Cells To Colonize Human and Murine Enteroids.

Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids-cultured three-dimensional replicas of the mammalian gut-offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.

High-Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium.

Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study, e.g., microbe motility, binding and invasion of host cells, and intrahost-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging but poorly reflects the host tissue's physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms. IMPORTANCE Knowledge of dynamic niche-specific interactions between single microbes and host cells is essential to understand infectious disease progression. However, advances in this field have been hampered by the inherent conflict between the technical requirements for high-resolution live-cell imaging on the one hand and conditions that best mimic physiological infection niche parameters on the other. Toward bridging this divide, we present a methodology for differential interference contrast (DIC) imaging of pathogen interactions at the apical surface of enteroid-derived intestinal epithelia, providing both high spatial and temporal resolution. This alleviates the need for fluorescent reporters in live-cell imaging and provides dynamic information about microbe interactions with a nontransformed, confluent, polarized, and microvilliated human gut epithelium. Using this methodology, we uncover previously unrecognized stages of Salmonella and Giardia infection cycles at the epithelial surface.

Metal Oxide Nanolayer-Decorated Epitaxial Graphene: A Gas Sensor Study.

In this manuscript, we explore the sensor properties of epitaxially grown graphene on silicon carbide decorated with nanolayers of CuO, Fe3O4, V2O5, or ZrO2. The sensor devices were investigated in regard to their response towards NH3 as a typical reducing gas and CO, C6H6, CH2O, and NO2 as gases of interest for air quality monitoring. Moreover, the impact of operating temperature, relative humidity, and additional UV irradiation as changes in the sensing environment have been explored towards their impact on sensing properties. Finally, a cross-laboratory study is presented, supporting stable sensor responses, and the final data is merged into a simplified sensor array. This study shows that sensors can be tailored not only by using different materials but also by applying different working conditions, according to the requirements of certain applications. Lastly, a combination of several different sensors into a sensor array leads to a well-performing sensor system that, with further development, could be suitable for several applications where there is no solution on the market today.

Streptococcal sagA activates a proinflammatory response in mast cells by a sublytic mechanism.

Mast cells are implicated in the innate proinflammatory immune defence against bacterial insult, but the mechanisms through which mast cells respond to bacterial encounter are poorly defined. Here, we addressed this issue and show that mast cells respond vividly to wild type Streptococcus equi by up-regulating a panel of proinflammatory genes and by secreting proinflammatory cytokines. However, this response was completely abrogated when the bacteria lacked expression of sagA, whereas the lack of a range of other potential virulence genes (seeH, seeI, seeL, seeM, hasA, seM, aroB, pyrC, and recA) had no effect on the amplitude of the mast cell responses. The sagA gene encodes streptolysin S, a lytic toxin, and we next showed that the wild type strain but not a sagA-deficient mutant induced lysis of mast cells. To investigate whether host cell membrane perturbation per se could play a role in the activation of the proinflammatory response, we evaluated the effects of detergent- and pneumolysin-dependent lysis on mast cells. Indeed, exposure of mast cells to sublytic concentrations of all these agents resulted in cytokine responses of similar amplitudes as those caused by wild type streptococci. This suggests that sublytic membrane perturbation is sufficient to trigger full-blown proinflammatory signalling in mast cells. Subsequent analysis showed that the p38 and Erk1/2 signalling pathways had central roles in the proinflammatory response of mast cells challenged by either sagA-expressing streptococci or detergent. Altogether, these findings suggest that sagA-dependent mast cell membrane perturbation is a mechanism capable of activating the innate immune response upon bacterial challenge.

Epitaxial Graphene Sensors Combined with 3D-Printed Microfluidic Chip for Heavy Metals Detection.

In this work, we investigated the sensing performance of epitaxial graphene on Si-face 4H-SiC (EG/SiC) for liquid-phase detection of heavy metals (e.g., Pb and Cd), showing fast and stable response and low detection limit. The sensing platform proposed includes 3D-printed microfluidic devices, which incorporate all features required to connect and execute lab-on-chip (LOC) functions. The obtained results indicate that EG exhibits excellent sensing activity towards Pb and Cd ions. Several concentrations of Pb2+ solutions, ranging from 125 nM to 500 µM, were analyzed showing Langmuir correlation between signal and Pb2+ concentrations, good stability, and reproducibility over time. Upon the simultaneous presence of both metals, sensor response is dominated by Pb2+ rather than Cd2+ ions. To explain the sensing mechanisms and difference in adsorption behavior of Pb2+ and Cd2+ ions on EG in water-based solutions, we performed van-der-Waals (vdW)-corrected density functional theory (DFT) calculations and non-covalent interaction (NCI) analysis, extended charge decomposition analysis (ECDA), and topological analysis. We demonstrated that Pb2+ and Cd2+ ions act as electron-acceptors, enhancing hole conductivity of EG, due to charge transfer from graphene to metal ions, and Pb2+ ions have preferential ability to binding with graphene over cadmium. Electrochemical measurements confirmed the conductometric results, which additionally indicate that EG is more sensitive to lead than to cadmium.

Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production.

Mast cells contribute to the pathology of allergic and other disorders. Strategies to interfere with harmful mast cell-related activities are therefore warranted. Previously we established a principle for inducing selective apoptosis of mast cells, by the use of lysosomotropic agents that cause secretory granule permeabilization, leading to production of reactive oxygen species (ROS). However, the mechanism of ROS production has not been known. Here we addressed this issue. Live microscopy analysis showed that the secretory granules comprise major subcellular compartments for ROS production in response to mefloquine. As further signs for the primary involvement of secretory granules, both ROS production and cell death was blunted in mast cells lacking serglycin, a secretory granule-restricted proteoglycan. Inhibition of granule acidification caused an essentially complete blockade of granule permeabilization, ROS production and cell death in response to mefloquine. ROS production was also attenuated in the presence of an iron chelator, and after inhibition of either granzyme B or the ERK1/2 MAP kinase signaling pathway. Together, our findings reveal that the mast cell secretory granules constitute major sites for ROS production in mast cells subjected to lysosomotropic challenge. Moreover, this study reveals a central role for granule acidification in ROS generation and the pro-apoptotic response triggered downstream of secretory granule permeabilization.