Cell Population Level Research Articles

Integrin α3β1 signaling in regulation of the SK-Mel-147 melanoma cell senescence

Using a model of the human SK-Mel-147 melanoma cell line, it was shown that blocking the expression of integrin α3β1 by transduction of cells with α3-specific shRNA did not affect their proliferation, but sharply increased the proportion of SA-β-Gal-positive cells, a phenotypic feature of cell senescence. These findings were accompanied by a significant increase in the activity of the Akt and mTOR protein kinases and the expression of p53 and p21 oncosupressors. Pharmacological inhibition of mTORC1 reduced the number SA-β-Gal-positive cells in the SK-Mel-147 cell population depleted of α3β1. Based on our recent data on a non-canonical function of Akt isomers in the regulation of SK-Mel-147 cell senescence caused by deficiency of α2β1 receptor, we investigated the role of Akt isomers in senescence induced by the α3β1 knockdown. It appeared that in the cell population with downregulated α3β1, inhibition of Akt1 reduced the number SA-β-Gal positive cells to the level of control cell population, while inhibition of Akt2 had no visible effect. Our results demonstrate that the laminin-specific integrin α3β1, like the collagen-specific receptor α2β1, is involved in tumor cell protection from senescence, and senescence induced by α3β1 depletion, like that caused by α2β1 deficiency, is based on a signaling mechanism employing a non-canonical function of the Akt1 isoform.

A bioinformatics approach to introduce novel multi-epitope vaccines against Acinetobacter baumannii retrieved from immunogenic extracellular loops of outer membrane proteins

Increasing antibiotic resistance of Acinetobacter baumannii highlights the urgent need to develop an effective vaccine to combat it. Despite all efforts, no approved vaccine against this bacterium is on the market, and attempts are underway to find effective vaccines. Due to their surface exposure, outer membrane proteins (OMPs) are excellent vaccine targets. The extracellular loops of OMPs are highly immunogenic. Therefore, in the present study, we aimed to detect the conserved, exposed, and immunogenic linear B-cell epitopes among sixty-five OMPs available in 35 A. baumannii genomes deposited in the Vaxign database. According to defined criteria (allergenicity, antigenicity, and non-similarity to host), four linear B-cell epitopes were selected as highly immunogenic peptides. Epitope shuffling was performed to select the best arrangements of these epitopes. We proposed six different multi-epitope vaccines consisting of two Naked arrangements, two loop-less C-lobe (LCL) structures, and two FliC-based constructs. An LCL-based multi-epitope vaccine showed the best molecular docking score with human TLRs (TLR 1, 2, 4, and 5) and immune simulation results (Th1 cell population, IFN-γ, IL-12, IgM, IgG1, and IgG2 levels). This study provided a valuable library of exposed epitopes including 26 conserved, linear and immunogenic B-cell epitopes (belonged to 14 OMPs), and three optimized multi-epitope vaccines. However, more experimental studies are needed to confirm the actual immunoreactivity of these vaccine candidates.

Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata).

Primordial germ cells (PGCs) are undifferentiated gametes with heterogeneity, an evolutionarily conserved characteristic across various organisms. Although dynamic selection at the level of early germ cell populations is an important biological feature linked to fertility, the heterogeneity of PGCs in avian species has not been characterized. In this study, we sought to evaluate PGC heterogeneity in zebra finch using a single-cell RNA sequencing (scRNA-seq) approach. Using scRNA-seq of embryonic gonadal cells from male and female zebra finches at Hamburger and Hamilton (HH) stage 28, we annotated nine cell types from 20 cell clusters. We found that PGCs previously considered a single population can be separated into three subtypes showing differences in apoptosis, proliferation, and other biological processes. The three PGC subtypes were specifically enriched for genes showing expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Additionally, we discovered a novel biomarker, SMC1B, for gonadal PGCs in zebra finch. The results provide the first evidence of substantial heterogeneity in PGCs previously considered a single population in birds. This discovery expands our understanding of PGCs to avian species, and provides a basis for further research.

Effects of BCL11A Shmir-Induced Post-Transcriptional Silencing on Hemoglobin Polymer Inhibition in Single Red Blood Cells at Physiologic Oxygen Tension

IntroductionFetal hemoglobin (HbF) induction is a successful strategy for treating sickle cell disease. Higher levels of total HbF% are associated with better clinical outcomes, and a more uniform distribution of HbF across individual red blood cells (RBCs) is predicted to lead to better outcomes by inhibiting hemoglobin (Hb) polymerization in a higher fraction of individual RBCs. 1 Post-transcriptional silencing of BCL11A using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression has been safe and demonstrated high levels of total HbF expression broadly distributed in red cells in a pilot clinical study (NCT 03282656). 2 We assessed the effects of BCL11A down-regulation on Hb polymer formation at the single-RBC level in 7 subjects in this trial (BCL) using novel assays of single-RBC Hb polymer content levels and single-RBC HbF.MethodsHb polymer was detected in individual RBCs using a previously published microfluidic system. 3 The system detects Hb polymer based on RBC morphology and on the principle that Hb polymer lowers RBC oxygen saturation in proportion to polymer concentration. 4,5 Single-RBC HbF mass was measured by flow cytometry using a previously published protocol. 6 These two measurements of distributions of single-RBC HbF mass and single-RBC Hb polymer were then integrated at the cell-population level to estimate the threshold of single-RBC HbF needed to inhibit formation of a detectable level of polymer as function of oxygen tension. Blood samples from BCL subjects (HbF% range 23 - 44, after stabilizing post-therapy) were compared to 14 hydroxyurea (HU) responsive patients (mean HbF% = 17, range 6 - 32), including 3 highly-responsive patients (HbF% = 25, 27, 31) as well as 4 individuals with sickle cell trait (AS).ResultsAt 3.7% oxygen tension typical of the human microcirculation 7 and some regions of the central nervous system, 7 the typical BCL subject had detectable levels of Hb polymer in 52% (median) of RBCs vs. 66% for HU (p = 0.02) and 6% for AS (Figure 1A). At lower oxygen tensions of 1.7% typical of the bone marrow 7 or kidney medulla 7 BCL subjects had a polymerized RBC fraction of 68%, compared to 79% for HU (p<0.001) and 30% for AS. Fractions of RBC with polymer in BCL subjects stabilized about 3-6 months after treatment. By combining measurements of single-RBC HbF mass and single-RBC polymer at the population level, we estimated the threshold HbF mass or fraction required to inhibit detectable levels of polymer as a function of oxygen tension. We compared single-RBC HbF thresholds for the BCL cohort to the three HU high responders. The mean HbF threshold for polymerization inhibition estimated for the BCL cohort was lower (18%) than that for the HU high responders (25%) but was not statistically significant (p = 0.07) in this initial analysis.ConclusionsBCL11A down-regulation in seven clinical trial subjects is associated with favorable distributions of single-RBC polymer at multiple physiologic oxygen tensions compared to HU. Future study is needed to determine whether these favorable Hb polymer distributions are simply a result of higher total HbF or also due to lower single-RBC HbF thresholds for polymer inhibition.

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture.

Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.

Cell-to-cell variability in JAK2/STAT5 pathway components and cytoplasmic volumes defines survival threshold in erythroid progenitor cells.

Survival or apoptosis is a binary decision in individual cells. However, at the cell-population level, a graded increase in survival of colony-forming unit-erythroid (CFU-E) cells is observed upon stimulation with erythropoietin (Epo). To identify components of Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signal transduction that contribute to the graded population response, we extended a cell-population-level model calibrated with experimental data to study the behavior in single cells. The single-cell model shows that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of Epo receptor (EpoR):JAK2 complexes and of SHP1, as well as the extent of nuclear import because of the large variance in the cytoplasmic volume of CFU-E cells. 24-118 pSTAT5 molecules in the nucleus for 120min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes is sufficient to convert a switch-like behavior at the single-cell level to a graded population-level response.

Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML

IntroductionAcute myeloid leukemia (AML) is a disease of myeloid lineage precursors with poor overall survival and limited treatment options in relapsed patients. Ex vivo drug testing is an approach to identify potential responders for novel targeted agents. However, AML bone marrow is comprised of a mixture of malignant and non-malignant cell types, and may contain myeloid cells at distinct differentiation stages. Here, we evaluated the ex vivo sensitivity of primary AML samples at a detailed cell-population level using a multicolor high-throughput flow cytometer (FACS).MethodsBone marrow (BM) mononuclear cells of 23 AML patients were plated on 96-well plates in mononuclear cell media (PromoCell). Seven FDA approved drugs were used, including signal transduction inhibitors that had shown AML specific responses in our earlier studies (Pemovska et al. Cancer Discovery 2013 ): trametinib, ruxolitinib, everolimus, sunitinib, venetoclax, plus standard of care drugs idarubicin and cytarabine. After 3-day incubation, cells were stained with six cell surface markers (CD45, CD34, CD33, CD14, CD123, CD38) to identify major cell populations present in many AML samples: blasts, promonocytes/monocytes, lymphocytes and granulopoietic cells. Flow cytometric analysis was performed using the iQue Screener PLUS instrument (Intellicyt). Apoptotic and dead cells were removed from the analysis (Annexin-V/7-AAD) and live cell counts of different cell populations after drug treatment were measured. For comparison, the viability of the samples was also assessed using the CellTiter-Glo (CTG) assay that uses the amount of ATP as an indicator of metabolically active cells.ResultsImmunophenotyping of the AML samples showed large inter-sample variation in cell composition. The BM blast cell population out of the CD45+ positive cells ranged between 17-94% and the non-malignant lymphocyte population ranged from 0.4 to 46%. High numbers of promonocytes/monocytes were observed in samples from FAB M4/5 patients as expected, while FAB M0/1/2 patient samples mainly consisted of blasts and lymphocytes. FACS analysis of cell population specific drug responses revealed that blast cells of different patients had highly different drug sensitivities whereas lymphocytes and granulocytes did not show differences in drug sensitivities between the AML samples (Figure 1A). Interestingly, promonocytes/monocytes that are mainly present in FAB M4/5 samples but also in small quantities in some FAB M0/1/2 samples were systematically more sensitive to MEK inhibition, but more resistant to BCL-2 inhibition when compared to blasts (Figure 1A-B). In line with this observation, the average overall BM cell sensitivity of myelomonocytic/monocytic (FAB M4/5) samples as measured by CTG showed decreased sensitivity to venetoclax (P<0.01) and increased sensitivity to trametinib (P<0.05) when compared to less mature (FAB M0/1/2) AMLs. Initial differential gene expression analysis revealed enrichment of genes in the RAS/MAPK pathway in samples diagnosed with FAB M4/5. Amongst the tested compounds, venetoclax showed the highest toxic effect against blast cells even though 40% of the samples were at least partly resistant. Intriguingly, we observed that by combining venetoclax (50nM) with trametinib (25nM) or ruxolitinib (300nM), chemo- and venetoclax-resistant blast cells were effectively killed ex vivo (>80%) in all tested samples.ConclusionFACS-based drug testing ex vivo enables accurate drug sensitivity profiling of AML blast cells even in samples with high numbers of more mature leukemic cells and healthy cells. Our data indicate heterogeneity in drug responses of AML cells at different stages of myeloid differentiation, with high sensitivity to BCL-2 inhibition and resistance to MEK inhibition of the primitive blasts compared to more mature monocytic cells. MEK inhibitor sensitivity in FAB M4/5 AMLs might be linked to the inherent characteristics of monocyte lineage cells. Furthermore, we show that venetoclax-resistant blast cells can be effectively targeted by combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib. [Display omitted] DisclosuresMustjoki:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Heckman:Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

The Effect of Probiotics on Health Outcomes in the Elderly: A Systematic Review of Randomized, Placebo-Controlled Studies.

Increasing evidence suggests that probiotic supplementation may be efficacious in counteracting age-related shifts in gut microbiota composition and diversity, thereby impacting health outcomes and promoting healthy aging. However, randomized controlled trials (RCTs) with probiotics in healthy older adults have utilized a wide variety of strains and focused on several different outcomes with conflicting results. Therefore, a systematic review was conducted to determine which outcomes have been investigated in randomized controlled trials with probiotic supplementation in healthy older adults and what has been the effect of these interventions. For inclusion, studies reporting on randomized controlled trials with probiotic and synbiotic supplements in healthy older adults (defined as minimum age of 60 years) were considered. Studies reporting clinical trials in specific patient groups or unhealthy participants were excluded. In addition to assessment of eligibility and data extraction, each study was examined for risk of bias and quality assessment was performed by two independent reviewers. Due to the heterogeneity of outcomes, strains, study design, duration, and methodology, we did not perform any meta-analyses and instead provided a narrative overview of the outcomes examined. Of 1997 potentially eligible publications, 17 studies were included in this review. The risk of bias was low, although several studies failed to adequately describe random sequence generation, allocation concealment, and blinding. The overall study quality was high; however, many studies did not include sample calculations, and the majority of studies had a small sample size. The main outcomes examined in the trials included microbiota composition, immune-related measurements, digestive health, general well-being, cognitive function, and lipid and other biomarkers. The most commonly assessed outcome with the most consistent effect was microbiota composition; all but one study with this outcome showed significant effects on gut microbiota composition in healthy older adults. Overall, probiotic supplementation had modest effects on markers of humoral immunity, immune cell population levels and activity, as well as the incidence and duration of the common cold and other infections with some conflicting results. Digestive health, general-well-being, cognitive function, and lipid and other biomarkers were investigated in a very small number of studies; therefore, the impact on these outcomes remains inconclusive. Probiotics appear to be efficacious in modifying gut microbiota composition in healthy older adults and have moderate effects on immune function. However, the effect of probiotic supplementation on other health outcomes remains inconclusive, highlighting the need for more well-designed, sufficiently-powered studies to investigate if and the mechanisms by which probiotics impact healthy aging.

Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival.

Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

Identification of Cell Types that Express Dio3 Deiodinase, a Thyroid Hormone-Inactivating Enzyme, Using a Dio3-CreERT2 Reporter System

Background: Thyroid hormone promotes development, growth and metabolism. The level of thyroid hormone ligand (triiodothyronine, T3) in tissues depends not only on circulating levels but also upon tight regulation by activating and inactivating deiodinases within tissues. Type 3 deiodinase (Dio3) inactivates T3 and its precursor thyroxine (T4) and mediates many functions including in neurodevelopmental, sensory and reproductive systems. Dio3 is subject to genomic imprinting. Despite its critical functions, Dio3 is often expressed transiently and at low levels in restricted cell populations making it difficult to detect in natural tissues. Methods: To visualize Dio3 expression at cellular resolution, we derived a Dio3-CreERt2 knockin allele that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio3 gene. When crossed with Ai6 reporter mice, Dio3-CreERt2-positive cells display fluorescent signals. When tamoxifen-treated at neonatal ages, Dio3-CreERt2 recapitulates endogenous Dio3 expression as previously reported in brain: in the bed nucleus of the stria terminalis and preoptic nuclei. In addition, we uncovered several positive cell groups in the hypothalamus, brain stem, pituitary and other tissues. Drastic differences were observed for Dio3-CreERt2 as a paternally versus maternally inherited allele, revealing imprinting effect in specific cell types. Dio3-CreERT2 activity is enhanced by T3 administration, in accordance with Dio3 as a T3-indicible gene. Conclusion: The Dio3-CreERT2 model sensitively reveals Dio3-expressing cell types in tissues. The model is useful for studying expression patterns, imprinting and lineage tracing of Dio3-positive cells during development and homeostatic challenges.

Transient cell stiffening triggered by magnetic nanoparticle exposure

BackgroundThe interactions between nanoparticles and the biological environment have long been studied, with toxicological assays being the most common experimental route. In parallel, recent growing evidence has brought into light the important role that cell mechanics play in numerous cell biological processes. However, despite the prevalence of nanotechnology applications in biology, and in particular the increased use of magnetic nanoparticles for cell therapy and imaging, the impact of nanoparticles on the cells’ mechanical properties remains poorly understood.ResultsHere, we used a parallel plate rheometer to measure the impact of magnetic nanoparticles on the viscoelastic modulus G*(f) of individual cells. We show how the active uptake of nanoparticles translates into cell stiffening in a short time scale (< 30 min), at the single cell level. The cell stiffening effect is however less marked at the cell population level, when the cells are pre-labeled under a longer incubation time (2 h) with nanoparticles. 24 h later, the stiffening effect is no more present. Imaging of the nanoparticle uptake reveals almost immediate (within minutes) nanoparticle aggregation at the cell membrane, triggering early endocytosis, whereas nanoparticles are almost all confined in late or lysosomal endosomes after 2 h of uptake. Remarkably, this correlates well with the imaging of the actin cytoskeleton, with actin bundling being highly prevalent at early time points into the exposure to the nanoparticles, an effect that renormalizes after longer periods.ConclusionsOverall, this work evidences that magnetic nanoparticle internalization, coupled to cytoskeleton remodeling, contributes to a change in the cell mechanical properties within minutes of their initial contact, leading to an increase in cell rigidity. This effect appears to be transient, reduced after hours and disappearing 24 h after the internalization has taken place.

Simple Fluorogenic Cellular Assay for Histone Deacetylase Inhibitors Based on Split-Yellow Fluorescent Protein and Intrabodies.

Histone deacetylase (HDAC) inhibitors that regulate the posttranslational modifications of histone tails are therapeutic drugs for many diseases such as cancers, neurodegenerative diseases, and asthma; however, convenient and sensitive methods to measure the effect of HDAC inhibitors in cultured mammalian cells remain limited. In this study, a fluorogenic assay was developed to detect the acetylation of lysine 9 on histone H3 (H3K9ac), which is involved in several cancers, Alzheimer’s disease, and autism spectrum disorder. To monitor the changes in H3K9ac levels, an H3K9ac-specific intrabody fused with a small fragment FP11 of the split-yellow fluorescent protein (YFP) (scFv-FP11) was expressed in mammalian cells, together with a larger YFP fragment FP1-10 fused with a nuclear localization signal. When the intranuclear level of H3K9ac is increased, the scFv-FP11 is more enriched in the nucleus via passive diffusion through the nuclear pores from the cytoplasm, which increases the chance of forming a fluorescent complex with the nuclear YFP1-10. The results showed that the YFP fluorescence increased when the cells were treated with HDAC inhibitors. Moreover, the sensitivity of the split YFP reporter system to three HDAC inhibitors was higher than that of a conventional cell viability test. The assay system will be a simple and sensitive detection method to evaluate HDAC inhibitor activities at the levels of both single cells and cell populations.

Single-cell transcriptomics provides new insights into the role of fibroblasts during peritoneal fibrosis.

BackgroundThe contributions of various types of cell populations in dialysis‐related peritoneal fibrosis are poorly understood. Single‐cell RNA sequencing brings single‐cell level resolution to the analysis of cellular transcriptomics, which provides a new way to further characterize the distinct roles and functional states of each cell population during peritoneal fibrosis.MethodsSingle‐cell transcriptomics from normal peritoneal tissues of six patients, from effluent of patients with short‐term peritoneal dialysis (less than 2 weeks, n = 6), and from long‐term peritoneal dialysis patients (more than 6 years, n = 4) were analyzed.ResultsWe identified a distinct cell component between samples among different groups. Functional analysis of the differentially expressed genes identified cell type specific biological processes relevant to different fibrosis stages. Well‐known key molecular mechanisms participating in the pathophysiology of peritoneal fibrosis were vitrified, and some of them were found to be restricted to specific cell types. Gradually growing enrichment of PI3K/AKT/mTOR pathway and impairment of oxidative phosphorylation in mesothelial cells and fibroblasts were found from healthy control, short‐term dialysis, to long‐term dialysis, respectively. The fibroblasts’ population obtained from the patients, who received peritoneal dialysis, showed a functional characteristic of immune‐chemotaxis and immune response, which was characterized by broadly significant increase in the expression of interleukins, chemokines, cytokines, and human leukocyte antigens. Furthermore, we described the intercellular crosstalk networks based on receptor‐ligand interactions, and highlighted a central role of fibroblasts in regulating the key mechanisms of peritoneal fibrosis through crosstalk with other cells.ConclusionsIn summary, despite describing information for fibrogenic molecular mechanisms in the resolution level of individual cell populations, this work identifies the significant functional evolution of fibroblasts during peritoneal fibrosis. This study also reveals the intercellular receptor‐ligand interactions in which the fibroblasts serve as a major node, eventually providing new insights into the role of fibroblasts during disease pathogenesis.

Oxytocin modulates neural processing of mitral/tufted cells in the olfactory bulb.

Oxytocin plays an important role in social recognition in rodents, which is mediated predominantly by the olfactory system. Although oxytocin modulates neural activity in the olfactory bulb, the underlying mechanism is largely unknown. Here, we studied how direct infusion of oxytocin into the olfactory bulb affect social interactions in mice and modulate the neural activity of mitral/tufted cells in the olfactory bulb. A three-chamber social interaction test was used in the behavioural test. For in vivo studies, single unit recordings, local field potential recordings and fibre photometry recordings were used to record the neural activity of olfactory bulb. For in vitro studies, we performed patch clamp recordings in the slice of the olfactory bulb. Behaviourally, direct oxytocin infusion in olfactory bulb increased performance in a social interaction task. Moreover, odour-evoked responses of mitral/tufted cells and neural discrimination of odours were both enhanced by oxytocin, whereas the spontaneous firing rate of mitral/tufted cells was reduced. At the neural network level, oxytocin decreased the amplitude of odour-evoked high gamma responses. At the cell population level, oxytocin decreased odour-evoked calcium responses (reflecting neural activity) specifically in granule cells. Moreover, in vitro slice recordings revealed that the inhibitory effect of oxytocin on mitral cell activity is mediated mainly by modulation of ATP-sensitive potassium channels and involves the oxytocin receptor-Gq-PLC-IP3 signalling pathway. Oxytocin modulates social interaction, likely by increasing the signal-to-noise ratio of odour responses in mitral cells which is partly through ATP-sensitive potassium channel.

Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures.

IntroductionIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.MethodsHere, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase.ResultsThe coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.ConclusionOur results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly.

Dissociation Constant of Integrin-RGD Binding in Live Cells from Automated Micropipette and Label-Free Optical Data.

The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant (Kd) of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of Kd2d = (4503 ± 1673) 1/µm2, a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a Kd within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy.

Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other in vitro methods.

IL-37 exerts therapeutic effects in experimental autoimmune encephalomyelitis through the receptor complex IL-1R5/IL-1R8.

Background: Interleukin 37 (IL-37), a member of IL-1 family, broadly suppresses inflammation in many pathological conditions by acting as a dual-function cytokine in that IL-37 signals via the extracellular receptor complex IL1-R5/IL-1R8, but it can also translocate to the nucleus. However, whether IL-37 exerts beneficial actions in neuroinflammatory diseases, such as multiple sclerosis, remains to be elucidated. Thus, the goals of the present study were to evaluate the therapeutic effects of IL-37 in a mouse model of multiple sclerosis, and if so, whether this is mediated via the extracellular receptor complex IL-1R5/IL-1R8.Methods: We used a murine model of MS, the experimental autoimmune encephalomyelitis (EAE). We induced EAE in three different single and double transgenic mice (hIL-37tg, IL-1R8 KO, hIL-37tg-IL-1R8 KO) and wild type littermates. We also induced EAE in C57Bl/6 mice and treated them with various forms of recombinant human IL-37 protein. Functional and histological techniques were used to assess locomotor deficits and demyelination. Luminex and flow cytometry analysis were done to assess the protein levels of pro-inflammatory cytokines and different immune cell populations, respectively. qPCRs were done to assess the expression of IL-37, IL-1R5 and IL-1R8 in the spinal cord of EAE, and in blood peripheral mononuclear cells and brain tissue samples of MS patients.Results: We demonstrate that IL-37 reduces inflammation and protects against neurological deficits and myelin loss in EAE mice by acting via IL1-R5/IL1-R8. We also reveal that administration of recombinant human IL-37 exerts therapeutic actions in EAE mice. We finally show that IL-37 transcripts are not up-regulated in peripheral blood mononuclear cells and in brain lesions of MS patients, despite the IL-1R5/IL-1R8 receptor complex is expressed.Conclusions: This study presents novel data indicating that IL-37 exerts therapeutic effects in EAE by acting through the extracellular receptor complex IL-1R5/IL-1R8, and that this protective physiological mechanism is defective in MS individuals. IL-37 may therefore represent a novel therapeutic avenue for the treatment of MS with great promising potential.

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection.

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.