Single-cell Activity Research Articles

Ganglion Cells with Sustained Activity in the Fish Retina and Their Possible Function in Evaluation of Visual Scenes

Background extracellular spike activity of single ganglion cells was recorded from axon terminals in the optic tectum of living immobilized fish. The sizes of the receptive fields of ON and OFF units with sustained responses (USR) amounted to 4–5° and were comparable with those of feature detectors. Generation of spike discharges by USR required contrast between the center and periphery of the receptive field. When there was no contrast, no spike activity appeared. The magnitude of the reaction was monotonically dependent on the level of this contrast. USR of both the ON and OFF types were connected with three types of cone (L, M, S). Both the center and periphery of the receptive field displayed color opponency, the center and periphery of the receptive field being opponent in terms of this characteristic. In other words, USR were double opponent and may thus take part in color discrimination. The simultaneous operation of feature detectors and ganglion cells with baseline activity separated into ON and OFF channels is represented retinotopically and may provide tectum opticum neurons with the visual scene information required for their function of controlling external attention.

A Sensorimotor Numerosity System.

Incoming sensory input provides information for the planning and execution of actions, which yield motor outcomes that are themselves sensory inputs. One dimension where action and perception strongly interact is numerosity perception. Many non-human animals can estimate approximately the number of external elements as well as their own actions, and neurons have been identified that respond to both. Recent psychophysical adaptation studies on humans also provide evidence for neural mechanisms responding to both the number of externally generated events and self-produced actions. Here we advance the idea that these strong connections may arise from dedicated sensorimotor mechanisms in the brain, part of a more generalized system interfacing action with the processing of other quantitative magnitudes such as space and time.

Comparative study of reverse flow activation and conventional activation with polymer electrolyte membrane fuel cell

Since fuel cell technology is in the commercialization stage, activation cost is also considered an important issue in addition to the manufacturing cost of fuel cell stack. As fuel cell capacity increases, activation time and required fuel consumption are issues that must be addressed to cut costs. In this context, this study suggests an activation method to reduce time and fuel consumption in polymer electrolyte membrane fuel cell. The effects of the newly suggested method, reverse-flow activation, is compared with the conventional activation method through experiments using single-cell and 5-cell stack. As a result, the suggested method reduced 33% of single-cell activation time and 43% of 5-cell stack activation time. A segmented fuel cell is introduced to inspect the local membrane activation progress in detail. As a result, the activation is accelerated in the reactant inlet region. When the reverse-flow activation is applied, it seems that the hydration of a membrane in advance near the reactant inlet region and the catalysts activated in advance near the reactant outlet region contribute to faster activation. As a result, the reverse-flow activation method reduces activation time and following fuel consumption.

Dual mechanisms of ictal high frequency oscillations in human rhythmic onset seizures

High frequency oscillations (HFOs) are bursts of neural activity in the range of 80 Hz or higher, recorded from intracranial electrodes during epileptiform discharges. HFOs are a proposed biomarker of epileptic brain tissue and may also be useful for seizure forecasting. Despite such clinical utility of HFOs, the spatial context and neuronal activity underlying these local field potential (LFP) events remains unclear. We sought to further understand the neuronal correlates of ictal high frequency LFPs using multielectrode array recordings in the human neocortex and mesial temporal lobe during rhythmic onset seizures. These multiscale recordings capture single cell, multiunit, and LFP activity from the human brain. We compare features of multiunit firing and high frequency LFP from microelectrodes and macroelectrodes during ictal discharges in both the seizure core and penumbra (spatial seizure domains defined by multiunit activity patterns). We report differences in spectral features, unit-local field potential coupling, and information theoretic characteristics of high frequency LFP before and after local seizure invasion. Furthermore, we tie these time-domain differences to spatial domains of seizures, showing that penumbral discharges are more broadly distributed and less useful for seizure localization. These results describe the neuronal and synaptic correlates of two types of pathological HFOs in humans and have important implications for clinical interpretation of rhythmic onset seizures.

Abstract PO-024: Tumor-specific cell populations in clear cell renal carcinoma associated with clinical outcome identified using single-cell protein activity inference

Abstract Primary Clear Cell Renal Carcinoma (ccRCC) is a highly heterogenous disease with a variable disease course post-surgery, and ccRCC tumor micro-environment has thus far not been characterized at the single-cell level with the whole-transcriptome resolution enabled by single-cell RNA Sequencing (scRNASeq). To elucidate the cellular and transcriptional mechanisms driving disease recurrence, we performed scRNASeq on both hematopoietic and non-hematopoietic populations from tumor and tumor-adjacent tissue from primary resections of treatment-naïve ccRCC patients (n=11), thus producing a complete atlas of the ccRCC tumor microenvironment. Furthermore, in order to mitigate gene dropout inherent to scRNASeq and commonly preventing detection of >80% of genes, we leveraged the VIPER algorithm, which quantitates protein activity, to infer the proteomic regulators of cell state. Clustering at the gene expression level enabled construction of lineage-specific gene regulatory networks applying ARACNe, the Algorithm for Reconstruction of Accurate Cellular Networks, from which protein activity of upstream regulatory molecules could be inferred by VIPER. Comparison with protein-level expression data from antibody-based, high-parameter spectral flow cytometry in the same patients shows that VIPER-measured protein activity systematically abrogated gene dropout effects on a repertoire of >6,000 regulatory proteins, comparing favorably with antibody-based measurements. This helped comprehensively characterize the individual cellular sub-populations comprising the ccRCC tumor and peri-tumor microenvironment, as well as their specific master regulators and candidate cell-cell interactions, revealing several populations undetectable by gene expression analysis. Specifically, we uncovered a novel tumor-specific, macrophage subpopulation characterized by significant upregulation of TREM2, APOE, and C1Q, as validated by spatially-resolved, quantitative multispectral immunofluorescence (qmIF). These tumor-specific macrophages were statistically significantly over-represented in a separate validation cohort of patients who recurred following surgery (n=4) compared to patients who did not recur (n=4). This study highlights the substantial resolution increase afforded by VIPER and identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential biomarker for ccRCC recurrence and a candidate target for intervention. Furthermore, it provides a highly-generalizable methodology to study the role of rare subpopulations by leveraging VIPER on scRNASeq data. Citation Format: Aleksandar Obradovic, Nivedita Showdhury, Casey Ager, Vinson Wang, Lukas Vlahos, Xinzheng V. Guo, David H. Aggen, James McKiernan, Andrea Califano, Charles G. Drake. Tumor-specific cell populations in clear cell renal carcinoma associated with clinical outcome identified using single-cell protein activity inference [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-024.

DIY enzyme labelled fluorescence alcohol (ELFA) standard production protocol to quantify single-cell phosphatase activity (SCPA) of microplankton

Extracellular enzyme activities (EEA) are crucial components of microbial food web interactions and biogeochemical cycles in aquatic ecosystems. They also represent relevant biological traits in the ecophysiology of phytoplankton and other components of microbial plankton. To assess species-specific and (sub-)population-level characteristics of phytoplankton EEA at the single-cell level and close-to-in-situ conditions solely the enzyme labelled fluorescence (ELF)-based substrates have been used, because they become fluorescent and precipitate around the enzyme activity location upon enzymatic cleavage. However, the enzyme-labelled fluorescence alcohol (ELFA) standard is no longer commercially available, hence standard curves cannot be run anymore and single-cell phosphatase activity (SCPA) is no longer quantifiable. Therefore, we introduce a simple protocol for an ELFA standard do it yourself (DIY) production to enable quantifying microplankton SCPA again. This protocol is based on fluorescence measurements easily available to environmental enzyme activity laboratories, and it circumvents any need for chemical synthesis equipment and knowledge. The method is based on a controlled reaction of the ELF-phosphate (ELFP) substrate with commercially available alkaline phosphatase, which efficiently turns all the substrate into ELFA product. The ELFA product was dried out and dissolved again in dimethyl sulfoxide (DMSO) for storage. The ELFA concentration of that standard-to-be ELFA solution in DMSO was determined by linear regression between a low concentration dilution series of ELFA solution measured fluorimetrically and parallel measurements of a series of phosphatase-catalysed reactions at an overlapping ELFP concentration range. Finally, the fluorescence- and concentration-stable ELFA solution in DMSO with a known concentration constitutes the ELFA standard that is necessary to quantify bulk (fluorimeter) and single-cell (microscope and flow cytometer) phosphatase activity in microplankton.

Single-cell activity in human STG during perception of phonemes is organized according to manner of articulation

One of the central tasks of the human auditory system is to extract sound features from incoming acoustic signals that are most critical for speech perception. Specifically, phonological features and phonemes are the building blocks for more complex linguistic entities, such as syllables, words and sentences. Previous ECoG and EEG studies showed that various regions in the superior temporal gyrus (STG) exhibit selective responses to specific phonological features. However, electrical activity recorded by ECoG or EEG grids reflects average responses of large neuronal populations and is therefore limited in providing insights into activity patterns of single neurons. Here, we recorded spiking activity from 45 units in the STG from six neurosurgical patients who performed a listening task with phoneme stimuli. Fourteen units showed significant responsiveness to the stimuli. Using a Naïve-Bayes model, we find that single-cell responses to phonemes are governed by manner-of-articulation features and are organized according to sonority with two main clusters for sonorants and obstruents. We further find that ‘neural similarity’ (i.e. the similarity of evoked spiking activity between pairs of phonemes) is comparable to the ‘perceptual similarity’ (i.e. to what extent two phonemes are judged as sounding similar) based on perceptual confusion, assessed behaviorally in healthy subjects. Thus, phonemes that were perceptually similar also had similar neural responses. Taken together, our findings indicate that manner-of-articulation is the dominant organization dimension of phoneme representations at the single-cell level, suggesting a remarkable consistency across levels of analyses, from the single neuron level to that of large neuronal populations and behavior.

Cell-Cycle-Dependent ERK Signaling Dynamics Direct Fate Specification in the Mammalian Preimplantation Embryo.

Despite the noisy nature of single cells, multicellular organisms robustly generate different cell types from one zygote. This process involves dynamic cross regulation between signaling and gene expression that is difficult to capture with fixed-cell approaches. To study signaling dynamics and fate specification during preimplantation development, we generated a transgenic mouse expressing the ERK kinase translocation reporter and measured ERK activity in single cells of live embryos. Our results show primarily active ERK in both the inner cell mass and trophectoderm cells due to fibroblast growth factor (FGF) signaling. Strikingly, a subset of mitotic events results in a short pulse of ERK inactivity in both daughter cells that correlates with elevated endpoint NANOG levels. Moreover, endogenous tagging of Nanog in embryonic stem cells reveals that ERK inhibition promotes enhanced stabilization of NANOG protein after mitosis. Our data show that cell cycle, signaling, and differentiation are coordinated during preimplantation development.

Single-Cell Activation of the cAMP-Signaling Pathway in 3D Tissues with FRET-Assisted Two-Photon Activation of bPAC.

Bacterial photoactivated adenylyl cyclase (bPAC) has been widely used in signal transduction research. However, due to its low two-photon absorption, bPAC cannot be efficiently activated by two-photon (2P) excitation. Taking advantage of the high two-photon absorption of monomeric teal fluorescent protein 1 (mTFP1), we herein developed 2P-activatable bPAC (2pabPAC), a fusion protein consisting of bPAC and mTFP1. In 2pabPAC, the energy absorbed by mTFP1 excites bPAC by Fürster resonance energy transfer (FRET) at ca. 43% efficiency. The light-induced increase in cAMP was monitored by a red-shifted FRET biosensor for PKA. In 3D MDCK cells and mouse liver, PKA was activated at single-cell resolution under a 2P microscope. We found that PKA activation in a single hepatocyte caused PKA activation in neighboring cells, indicating the propagation of PKA activation. Thus, 2pabPAC will provide a versatile platform for controlling the cAMP signaling pathway and investigating cell-to-cell communication in vivo.

Silicon Photomultipliers as a Low-Cost Fluorescence Detector for Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation method capable of handling small sample volumes (∼pL) and low (∼yoctomole) detection limits and, as such, is ideal for applications that require high sensitivity, such as single-cell analysis (Chen et al. Anal. Chem. 1996, 68 (4), 690-696; Cohen et al. Annu. Rev. Anal. Chem. 2008, 1 (1), 165-190; Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751). Low-cost CE instrumentation is quickly expanding, but low-cost, open-source fluorescence detectors with ultrasensitive detection limits are lacking (Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751; Fang et al. Electrophoresis 2016, 37 (17-18), 2376-2383; Casto et al. Anal. Chem. 2019, 40 (1), 65-78). Silicon photomultipliers (SiPM) are inexpensive, low-footprint detectors with the potential to fill the role as a detector when cost, size, and customization are important. In this work, we demonstrate the use of a SiPM in CE with zeptomolar detection limits and a dynamic range spanning 5 orders of magnitude, comparable to photomultiplier detectors. The performance of these detectors was measured using a continuous wave excitation laser in an epifluorescence detection configuration. We characterize the performance of the SiPM as a highly sensitive detector by measuring enzyme activity in single cells. This simple, small footprint, and low-cost (<$130) light detection circuit will be beneficial for open-source, portable, and budget-friendly instrumentation requiring high sensitivity.

Reduced firing rates of pyramidal cells in the frontal cortex of APP/PS1 can be restored by acute treatment with levetiracetam

In recent years, aberrant neural oscillations in various cortical areas have emerged as a common physiological hallmark across mouse models of amyloid pathology and patients with Alzheimer's disease. However, much less is known about the underlying effect of amyloid pathology on single cell activity. Here, we used high-density silicon probe recordings from frontal cortex area of 9-month-old APP/PS1 mice to show that local field potential power in the theta and beta band is increased in transgenic animals, whereas single-cell firing rates, specifically of putative pyramidal cells, are significantly reduced. At the same time, these sparsely firing pyramidal cells phase-lock their spiking activity more strongly to the ongoing theta and beta rhythms. Furthermore, we demonstrated that the antiepileptic drug, levetiracetam, counteracts these effects by increasing pyramidal cell firing rates in APP/PS1 mice and uncoupling pyramidal cells and interneurons. Overall, our results highlight reduced firing rates of cortical pyramidal cells as a pathophysiological phenotype in APP/PS1 mice and indicate a potentially beneficial effect of acute levetiracetam treatment.

Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance

Human fungal infections may fail to respond to contemporary antifungal therapies in vivo despite in vitro fungal isolate drug susceptibility. Such a discrepancy between in vitro antimicrobial susceptibility and in vivo treatment outcomes is partially explained by microbes adopting a drug-resistant biofilm mode of growth during infection. The filamentous fungal pathogen Aspergillus fumigatus forms biofilms in vivo, and during biofilm growth it has reduced susceptibility to all three classes of contemporary antifungal drugs. Specific features of filamentous fungal biofilms that drive antifungal drug resistance remain largely unknown. In this study, we applied a fluorescence microscopy approach coupled with transcriptional bioreporters to define spatial and temporal oxygen gradients and single-cell metabolic activity within A. fumigatus biofilms. Oxygen gradients inevitably arise during A. fumigatus biofilm maturation and are both critical for, and the result of, A. fumigatus late-stage biofilm architecture. We observe that these self-induced hypoxic microenvironments not only contribute to filamentous fungal biofilm maturation but also drive resistance to antifungal treatment. Decreasing oxygen levels toward the base of A. fumigatus biofilms increases antifungal drug resistance. Our results define a previously unknown mechanistic link between filamentous fungal biofilm physiology and contemporary antifungal drug resistance. Moreover, we demonstrate that drug resistance mediated by dynamic oxygen gradients, found in many bacterial biofilms, also extends to the fungal kingdom. The conservation of hypoxic drug-resistant niches in bacterial and fungal biofilms is thus a promising target for improving antimicrobial therapy efficacy.

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This "wedge slice" was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Enhancement of cell growth by uncoupling extracellular electron uptake and oxidative stress production in sediment sulfate-reducing bacteria

Microbial extracellular electron uptake (EEU) from solid electron donors has critical implications for microbial energy acquisition in energy-limited environments as well as electrochemical microbial technologies. Although EEU supplies sufficient energy to support cellular growth, additional soluble electron donors are required for most microbes to grow on electrode surfaces. Here, we demonstrated that the minimization of exogenous and endogenous oxidative stress greatly enhanced the growth rate of the sediment EEU-capable sulfate-reducing bacterium Desulfovibrio ferrophilus IS5 on an electrode without the addition of a soluble electron donor. Single-cell activity analysis by nanoscale secondary ion mass spectrometry showed that the metabolic activity of IS5 cells on the electrode was significantly enhanced following incubation in an H-type reactor, which was configured to reduce the exposure of cells to the potential oxidative stress source of the Pt counter electrode (CE). Additionally, the highest metabolic activity was observed at an electrode potential of −0.4 V (versus the standard hydrogen electrode), where electron uptake rate was not at peak. Compared to a single-chamber reactor, incubation in an H-type reactor at −0.4 V shortened the cell doubling time by 50-fold, which resulted in sufficient anabolism for cell replication (15N/Ntotal > 50%). The production of strongly oxidizing species at the CE was confirmed by X-ray photoelectron spectroscopy and inductively coupled plasma mass spectrometry analyses. Transcriptome analysis revealed overexpression of antioxidative genes in cells incubated at a potential with higher current production. These results suggested that higher levels of endogenous oxidative species were produced by a more reduced electron-transport chain from trace amounts of oxygen in the reactor, thereby lowering cell activity. In conclusion, EEU may enable sediment microbes to undergo enhanced cell growth and to find niches on minerals under anaerobic energy-limited conditions, where oxidative stress is much less likely to be present.

Pre-existing Cell States Control Heterogeneity of Both EGFR and CXCR4 Signaling.

CXCR4 and epidermal growth factor receptor (EGFR) represent two major families of receptors, G-protein coupled receptors and receptor tyrosine kinases, with central functions in cancer. While utilizing different upstream signaling molecules, both CXCR4 and EGFR activate kinases ERK and Akt, although single-cell activation of these kinases is markedly heterogeneous. One hypothesis regarding the origin of signaling heterogeneity proposes that intercellular variations arise from differences in pre-existing intracellular states set by extrinsic noise. While pre-existing cell states vary among cells, each pre-existing state defines deterministic signaling outputs to downstream effectors. Understanding causes of signaling heterogeneity will inform treatment of cancers with drugs targeting drivers of oncogenic signaling. We built a single-cell computational model to predict Akt and ERK responses to CXCR4- and EGFR-mediated stimulation. We investigated signaling heterogeneity through these receptors and tested model predictions using quantitative, live-cell time-lapse imaging. We show that the pre-existing cell state predicts single-cell signaling through both CXCR4 and EGFR. Computational modeling reveals that the same set of pre-existing cell states explains signaling heterogeneity through both EGFR and CXCR4 at multiple doses of ligands and in two different breast cancer cell lines. The model also predicts how phosphatidylinositol-3-kinase (PI3K) targeted therapies potentiate ERK signaling in certain breast cancer cells and that low level, combined inhibition of MEK and PI3K ablates potentiated ERK signaling. Our data demonstrate that a conserved motif exists for EGFR and CXCR4 signaling and suggest potential clinical utility of the computational model to optimize therapy.

Hybrid Electro-Plasmonic Neural Stimulation with Visible-Light-Sensitive Gold Nanoparticles.

Biomedical prosthetics utilizing electrical stimulation have limited, effective spatial resolution due to spread of electrical currents to surrounding tissue, causing nonselective stimulation. So, precise spatial resolution is not possible for traditional neural prosthetic devices, such as cochlear implants. More recently, alternative methods utilize optical stimulation, mainly infrared, sometimes paired with nanotechnology for stimulating action potentials. Infrared stimulation has its own drawbacks, as it may cause collateral heating of surrounding tissue. In previous work, we employed a plasmonic method for stimulation of an electrically excitable neuroblastoma cell line, which had limited success. Here, we report the development of a hybrid electro-plasmonic stimulation platform for spatially and temporally precise neural excitation to address the above deficiencies. Primary trigeminal neurons were costimulated in vitro in a whole-cell patch-clamp configuration with subthreshold-level short-duration (1-5 ms) electrical and visible light pulses (1-5 ms). The visible light pulses were aimed at a gold-nanoparticle-coated nanoelectrode placed alongside the neuron, within 2 μm distance. Membrane action potentials were recorded with a 3-fold higher success rate and 5-fold better poststimulation cell recovery rate than with pure optical stimulation alone. Also, electrical stimulus current input was being reduced by up to 40%. The subthreshold levels of electrical stimuli in conjunction with visible light (532 nm) reliably triggered trains of action potentials. This single-cell hybrid activation was reliable and repeatable, without any damage as observed with pure optical stimulation. This work represents an empirical cellular study of the membrane action potential response produced by the cultured primary sensory trigeminal neurons when costimulated with plasmonic and electrical (hybrid) stimulation. Our hybrid neurostimulation method can be used toward development of high-acuity neural modulation prosthetic devices, tunable for individual needs, which would qualify as a preferred alternative over traditional electrical stimulation technologies.

Calcium inhibitor inhibits high glucose‑induced hypertrophy of H9C2 cells.

The aim of the present study was to explore whether the hypertrophy of H9C2 cardiomyocytes was induced by high glucose, to investigate whether the calcium channel inhibitor (Norvasc) could inhibit this process and to clarify the possible signaling pathways. The morphology of H9C2 cells was observed under an optical microscope, and the cell surface area was measured by Image Pro Plus 6.1 software. Furthermore, fluorescence spectrophotometry was used to detect intracellular calcium concentration ([Ca2+]i). ELISA was performed to detect calcineurin (CaN) activity; reverse transcription-quantitative PCR and western blotting were performed to detect the mRNA and protein expression levels of CaN Aβ subunit (CnAβ), nuclear factor of activated T cells 3 (NFAT3) and β type myosin heavy chain (β-MHC). Cell size was increased with the increase in glucose concentration of culture medium at 48 and 72 h, respectively, and decreased with the addition of Norvasc compared with those without Norvasc (P<0.05). There was no significant difference in cell size with the addition of Norvasc compared with cells cultured with 5 mM glucose (P>0.05). The average [Ca2+]i activity of single cells in the 48- and 72-h culture groups treated with 50 mM glucose was significantly higher than cells treated with 5 mM glucose (P<0.05); and the fluorescent value of average [Ca2+]i activity of single cells was lower, following the addition of Norvasc than that without Norvasc (P<0.05). CaN activity in the 48- and 72-h culture group treated with 50 mM glucose was markedly higher than that treated with 5 mM glucose, and the activity of CaN notably decreased with the addition of Norvasc compared with those without Norvasc. The mRNA and protein expression levels of CnAβ, NFAT3 and β-MHC in the 48- and 72-h culture groups treated with 50 mM glucose were all significantly higher than those treated with 5 mM glucose (P<0.05). The mRNA and protein expression of CnAβ, NFAT3 and β-MHC cultured with 50 mM glucose were significantly decreased following the addition of Norvasc (P<0.05). Thus, the calcium channel inhibitor Norvasc may inhibit high glucose-induced hypertrophy of H9C2 cardiomyocytes by inhibiting the Ca2+-CaN-NFAT3 signaling pathway.

Continuous microfluidic encapsulation of single mesenchymal stem cells using alginate microgels as injectable fillers for bone regeneration

The encapsulation of cells in microscale hydrogels can provide a mimic of a three-dimensional (3D) microenvironment to support cell viability and functions and to protect cells from the environmental stress, which have been widely used in tissue regeneration and cell therapies. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. This microfluidic technique integrated on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process, which enables scalable cell encapsulation while retaining the viability and functionality of loaded cells. Remarkably, we observed MSCs encapsulated in Ca-alginate microgels at the single-cell level showed significantly enhanced osteogenesis and accelerated mineralization of the microgels which occurred only after 7 days of induction. Furthermore, MSCs laden in alginate microgels displayed significantly enhanced bone formation compared to MSCs mixed with microgels and acellular microgels in a rat tibial ablation model. To conclude, the current microfluidic technique represents a significant step toward continuous single cell encapsulation, fabrication, and purification. These microgels can boost bone regeneration by providing a controlled osteogenic microenvironment for encapsulated MSCs and facilitate stem cell therapy in the treatment of bone defects in a minimally invasive delivery way. Statement of SignificanceThe biological functions and therapeutic activities of single cells laden in microgels for tissue engineering remains less investigated. Here, we reported a microfluidic-based method for continuous encapsulation of single MSCs with high viability and functionality by integrating on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process. More importantly, MSCs encapsulated in alginate microgels at the single-cell level showed significantly enhanced osteogenesis, remarkably accelerated mineralization in vitro and bone formation capacity in vivo. Therefore, this single-cell encapsulation technique can facilitate stem cell therapy for bone regeneration and be potentially used in a variety of tissue engineering applications.

Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy.

Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this, we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients, which correlated with poor clinical response, and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL, MERTK, and their ligand GAS6. In xenograft tumors, intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs, revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling, we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus, bypass signaling can reciprocally amplify across nearby cell types, offering new opportunities for therapeutic design.

Assessing Cell Activities rather than Identities to Interpret Intra-Tumor Phenotypic Diversity and Its Dynamics

SummaryDespite advances in single-cell and molecular techniques, it is still unclear how to best quantify phenotypic heterogeneity in cancer cells that evolved beyond normal, known classifications. We present an approach to phenotypically characterize cells based on their activities rather than static classifications. We validated the detectability of specific activities (epithelial-mesenchymal transition, glycolysis) in single cells, using targeted RT-qPCR analyses and in vitro inductions. We analyzed 50 established activity signatures as a basis for phenotypic description in public data and computed cell-cell distances in 28,513 cells from 85 patients and 8 public datasets. Despite not relying on any classification, our measure correlated with standard diversity indices in populations of known structure. We identified bottlenecks as phenotypic diversity reduced upon colorectal cancer initiation. This suggests that focusing on what cancer cells do rather than what they are can quantify phenotypic diversity in universal fashion, to better understand and predict intra-tumor heterogeneity dynamics.