Large Cell Populations Research Articles

Application of single-cell sequencing in human cancer.

Precision medicine is emerging as a cornerstone of future cancer care with the objective of providing targeted therapies based on the molecular phenotype of each individual patient. Traditional bulk-level molecular phenotyping of tumours leads to significant information loss, as the molecular profile represents an average phenotype over large numbers of cells, while cancer is a disease with inherent intra-tumour heterogeneity at the cellular level caused by several factors, including clonal evolution, tissue hierarchies, rare cells and dynamic cell states. Single-cell sequencing provides means to characterize heterogeneity in a large population of cells and opens up opportunity to determine key molecular properties that influence clinical outcomes, including prognosis and probability of treatment response. Single-cell sequencing methods are now reliable enough to be used in many research laboratories, and we are starting to see applications of these technologies for characterization of human primary cancer cells. In this review, we provide an overview of studies that have applied single-cell sequencing to characterize human cancers at the single-cell level, and we discuss some of the current challenges in the field.

Direct Recording of Dendrodendritic Excitation in the Olfactory Bulb: Divergent Properties of Local and External Glutamatergic Inputs Govern Synaptic Integration in Granule Cells.

The olfactory bulb contains excitatory principal cells (mitral and tufted cells) that project to cortical targets as well as inhibitory interneurons. How the local circuitry in this region facilitates odor-specific output is not known, but previous work suggests that GABAergic granule cells plays an important role, especially during fine odor discrimination. Principal cells interact with granule cells through reciprocal dendrodendritic connections that are poorly understood. While many studies examined the GABAergic output side of these reciprocal connections, little is known about how granule cells are excited. Only two previous studies reported monosynaptically coupled mitral/granule cell connections and neither attempted to determine the fundamental properties of these synapses. Using dual intracellular recordings and a custom-built loose-patch amplifier, we have recorded unitary granule cell EPSPs evoked in response to mitral cell action potentials in rat (both sexes) brain slices. We find that the unitary dendrodendritic input is relatively weak with highly variable release probability and short-term depression. In contrast with the weak dendrodendritic input, the facilitating cortical input to granule cells is more powerful and less variable. Our computational simulations suggest that dendrodendritic synaptic properties prevent individual principal cells from strongly depolarizing granule cells, which likely discharge in response to either concerted activity among a large proportion of inputs or coactivation of a smaller subset of local dendrodendritic inputs with coincidence excitation from olfactory cortex. This dual-pathway requirement likely enables the sparse mitral/granule cell interconnections to develop highly odor-specific responses that facilitate fine olfactory discrimination.SIGNIFICANCE STATEMENT The olfactory bulb plays a central role in converting broad, highly overlapping, sensory input patterns into odor-selective population responses. How this occurs is not known, but experimental and theoretical studies suggest that local inhibition often plays a central role. Very little is known about how the most common local interneuron subtype, the granule cell, is excited during odor processing beyond the unusual anatomical arraignment of the interconnections (reciprocal dendrodendritic synapses). Using paired recordings and two-photon imaging, we determined the properties of the primary input to granule cells for the first time and show that these connections bias interneurons to fire in response to spiking in large populations of principal cells rather than a small group of highly active cells.

ExVivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide.

A Diagnostic Surprise: Primary Hodgkin's Lymphoma of the Lung.

An 81-year-old male presented to the emergency room with a 3-month history of progressive shortness of breath, productive cough with white sputum, and generalized weakness with 10-pound weight loss in 2 months. On presentation, the patient was afebrile, with blood pressure of 93/55 mm Hg and oxy-hemoglobin saturation of 92% on 2 liters of oxygen via nasal cannula. Complete blood count with differential was significant for white count of 12 400/mL. Brain natriuretic peptide level was 454 ng/mL. Postero-anterior chest radiograph showed multiple round opacities in the lung fields. Computed tomography scan of the chest confirmed multiple round densities in both the lung fields along with mild mediastinal lymphadenopathy. Core needle biopsy was performed. Immunohistochemical stains were positive for CD30 and CD15 in a population of large atypical cells amid a background of CD3-positive nonneoplastic cells. These results were in support of the diagnosis of classical Hodgkin’s lymphoma of the lung with histological appearance confirming nodular sclerosis type. The patient was started on chemotherapy but was readmitted in 20 days for acute respiratory distress and suffered cardiac arrest and subsequently died. This case highlights the fact that although primary pulmonary Hodgkin’s lymphoma of the lung is a rare entity, it should be thought of as a differential while evaluating lung masses. In these cases, definite diagnosis can only be made by biopsy and histology. Early commencement of chemotherapy and regular follow-up with oncology is essential.

Optophysiology of cardiomyocytes: characterizing cellular motion with quantitative phase imaging.

Quantitative phase imaging enables precise characterization of cellular shape and motion. Variation of cell volume in populations of cardiomyocytes can help distinguish their types, while changes in optical thickness during beating cycle identify contraction and relaxation periods and elucidate cell dynamics. Parameters such as characteristic cycle shape, beating frequency, duration and regularity can be used to classify stem-cell derived cardiomyocytes according to their health and, potentially, cell type. Unlike classical patch-clamp based electrophysiological characterization of cardiomyocytes, this interferometric approach enables rapid and non-destructive analysis of large populations of cells, with longitudinal follow-up, and applications to tissue regeneration, personalized medicine, and drug testing.

Optophysiology of cardiomyocytes: characterizing cellular motion with quantitative phase imaging.

Quantitative phase imaging enables precise characterization of cellular shape and motion. Variation of cell volume in populations of cardiomyocytes can help distinguish their types, while changes in optical thickness during beating cycle identify contraction and relaxation periods and elucidate cell dynamics. Parameters such as characteristic cycle shape, beating frequency, duration and regularity can be used to classify stem-cell derived cardiomyocytes according to their health and, potentially, cell type. Unlike classical patch-clamp based electrophysiological characterization of cardiomyocytes, this interferometric approach enables rapid and non-destructive analysis of large populations of cells, with longitudinal follow-up, and applications to tissue regeneration, personalized medicine, and drug testing.

Effects of sugar concentration on mango wine composition fermented by Saccharomyces cerevisiae MERIT.ferm

SummaryThis study investigated the effect of initial sugar concentrations (°Brix of 17, 23 and 30) on mango wine composition fermented by Saccharomyces cerevisiae MERIT.ferm. It was found that growth rate and maximum cell population were inversely correlated with initial sugar levels with the fastest growth rate and largest cell population in the low sugar fermentation. However, the cell population in the low and medium sugar fermentation declined significantly (from 8.7 and 8.2 log to 2 and 2.8 log, respectively) relative to the high sugar fermentation in which cell populations remained stable upon reaching the stationary phase (7.7 log). Glycerol production increased with increasing sugar content in low (13.4 g L−1), medium (14.5 g L−1) and high (15.9 g L−1) sugar fermentation. In addition, high sugar fermentation had a negative impact on volatile production with significantly lower amounts of acetate esters (1.5 mg L−1) but more acetic acid (0.54 g L−1) compared to the low (5.0 mg L−1 and 0.44 g L−1, respectively) and medium (3.7 mg L−1 and 0.49 g L−1, respectively) sugar fermentations. Furthermore, volatiles especially terpene hydrocarbons (α‐caryophyllene was released) present in mango juice were significantly metabolised after fermentation, while numerous new volatile compounds (such as isobutyl alcohol, isoamyl alcohol, ethyl and acetate esters) were produced. Some terpene alcohols were released and converted into corresponding acetyl esters. This may indicate that the mango wines fermented with different levels of sugars would have different flavour aromas.

Forward and backward evolutionary processes and allele frequency spectrum in a cancer cell population

A cancer grows from a single cell, thereby constituting a large cell population. In this work, we are interested in how mutations accumulate in a cancer cell population. We provide a theoretical framework of the stochastic process in a cancer cell population and obtain near exact expressions of allele frequency spectrum or AFS (only continuous approximation is involved) from both forward and backward treatments under a simple setting; all cells undergo cell divisions and die at constant rates, b and d, respectively, such that the entire population grows exponentially. This setting means that once a parental cancer cell is established, in the following growth phase, all mutations are assumed to have no effect on b or d (i.e., neutral or passengers). Our theoretical results show that the difference from organismal population genetics is mainly in the coalescent time scale, and the mutation rate is defined per cell division, not per time unit (e.g., generation). Except for these two factors, the basic logic is very similar between organismal and cancer population genetics, indicating that a number of well established theories of organismal population genetics could be translated to cancer population genetics with simple modifications.

Deblurring Signal Network Dynamics.

To orchestrate the function and development of multicellular organisms, cells integrate intra- and extracellular information. This information is processed via signal networks in space and time, steering dynamic changes in cellular structure and function. Defects in those signal networks can lead to developmental disorders or cancer. However, experimental analysis of signal networks is challenging as their state changes dynamically and differs between individual cells. Thus, causal relationships between network components are blurred if lysates from large cell populations are analyzed. To directly study causal relationships, perturbations that target specific components have to be combined with measurements of cellular responses within individual cells. However, using standard single-cell techniques, the number of signal activities that can be monitored simultaneously is limited. Furthermore, diffusion of signal network components limits the spatial precision of perturbations, which blurs the analysis of spatiotemporal processing in signal networks. Hybrid strategies based on optogenetics, surface patterning, chemical tools, and protein design can overcome those limitations and thereby sharpen our view into the dynamic spatiotemporal state of signal networks and enable unique insights into the mechanisms that control cellular function in space and time.

Transient expression of ZBTB32 in anti-viral CD8+ T cells limits the magnitude of the effector response and the generation of memory.

Virus infections induce CD8+ T cell responses comprised of a large population of terminal effector cells and a smaller subset of long-lived memory cells. The transcription factors regulating the relative expansion versus the long-term survival potential of anti-viral CD8+ T cells are not completely understood. We identified ZBTB32 as a transcription factor that is transiently expressed in effector CD8+ T cells. After acute virus infection, CD8+ T cells deficient in ZBTB32 showed enhanced virus-specific CD8+ T cell responses, and generated increased numbers of virus-specific memory cells; in contrast, persistent expression of ZBTB32 suppressed memory cell formation. The dysregulation of CD8+ T cell responses in the absence of ZBTB32 was catastrophic, as Zbtb32-/- mice succumbed to a systemic viral infection and showed evidence of severe lung pathology. We found that ZBTB32 and Blimp-1 were co-expressed following CD8+ T cell activation, bound to each other, and cooperatively regulated Blimp-1 target genes Eomes and Cd27. These findings demonstrate that ZBTB32 is a key transcription factor in CD8+ effector T cells that is required for the balanced regulation of effector versus memory responses to infection.

Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging of Hypochlorous Acid in Living Cells and Quantitative Application in Flow Cytometry.

Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system, and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective method for HClO detection in living systems, and most of fluorescent probes are mainly focused on cells imaging. Besides, accurate HClO quantitative information about individual cells in a large cell population is extremely important for understanding inflammation and cellular apoptosis as well. In our work, a turn-on fluorescent probe has been synthesized, which can selectively and sensitively detect HClO with fast response time. The probe is almost nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged C═N bonds which can undergo a nonradiative decay process in the excited state. Upon the addition of ClO-, the probe was oxidized to ring-opened fluorescent form and the fluorescence intensity was greatly enhanced. In live cell experiments, the probe was successfully applied to image exogenous ClO- in HeLa cells and endogenous HClO in RAW 264.7 macrophage cells. In particular, the quantitative information on exogenous and endogenous HClO can also be acquired in flow cytometry. Therefore, the probe not only can image exogenous and endogenous HClO but also provides a new and promising platform to quantitatively detect HClO in flow cytometry.

Nanoneedle-Based Sensing in Biological Systems.

Nanoneedles are high aspect ratio nanostructures with a unique biointerface. Thanks to their peculiar yet poorly understood interaction with cells, they very effectively sense intracellular conditions, typically with lower toxicity and perturbation than traditionally available probes. Through long-term, reversible interfacing with cells, nanoneedles can monitor biological functions over the course of several days. Their nanoscale dimension and the assembly into large-scale, ordered, dense arrays enable monitoring the functions of large cell populations, to provide functional maps with submicron spatial resolution. Intracellularly, they sense electrical activity of complex excitable networks, as well as concentration, function, and interaction of biomolecules in situ, while extracellularly they can measure the forces exerted by cells with piconewton detection limits, or efficiently sort rare cells based on their membrane receptors. Nanoneedles can investigate the function of many biological systems, ranging from cells, to biological fluids, to tissues and living organisms. This review examines the devices, strategies, and workflows developed to use nanoneedles for sensing in biological systems.

An image processing application for quantitative cross-correlative microscopy for large cell-populations: a gold nanoparticle radiosensitisation study

A robust analysis script was developed in MATLAB for cross-correlative quantification of internalised gold nanoparticle (AuNP) uptake in a large number of individual cells with the corresponding number of DNA double-strand breaks (DSBs) in the same cells. The correlation of inorganic NP content with a biological marker at the single-cell level will aid in the elucidation of mechanisms of NP radiosensitisation. PC-3 cells were co-cultured with AuNPs and irradiated using an iridium-192 source. AuNP uptake was measured using synchrotron X-ray fluorescence (XRF) and DSBs imaged via confocal microscopy. MATLAB 2016a was used to develop a script to cross-correlate the two imaging modalities and quantify both DSBs and internalised AuNP content in the same cell. Various user-defined options written into the script give a high degree of versatility, which can account for a large number of variables in experimental parameters and data acquisition. The analysis procedure is flexible and robust, which gives consistent consideration to the wide spectrum of potential input image/data sets. Quantitative correlative microscopy was achieved with a custom MATLAB script used to correlate γH2AX foci (a marker of DNA DSBs) from confocal microscopy with AuNP content acquired using synchrotron XRF at the single-cell level. The script can be extended to a broad range of multi-modality imaging spectroscopies.

BHLH proneural genes as cell fate determinants of entero-endocrine cells, an evolutionarily conserved lineage sharing a common root with sensory neurons

Entero-endocrine cells involved in the regulation of digestive function form a large and diverse cell population within the intestinal epithelium of all animals. Together with absorptive enterocytes and secretory gland cells, entero-endocrine cells are generated by the embryonic endoderm and, in the mature animal, from a pool of endoderm derived, self-renewing stem cells. Entero-endocrine cells share many structural/functional and developmental properties with sensory neurons, which hints at the possibility of an ancient evolutionary relationship between these two cell types. We will survey in this article recent findings that emphasize the similarities between entero-endocrine cells and sensory neurons in vertebrates and insects, for which a substantial volume of data pertaining to the entero-endocrine system has been compiled. We will then report new findings that shed light on the specification and morphogenesis of entero-endocrine cells in Drosophila. In this system, presumptive intestinal stem cells (pISCs), generated during early metamorphosis, undergo several rounds of mitosis that produce the endocrine cells and stem cells (ISCs) with which the fly is born. Clonal analysis demonstrated that individual pISCs can give rise to endocrine cells expressing different types of peptides. Immature endocrine cells start out as unpolarized cells located basally of the gut epithelium; they each extend an apical process into the epithelium which establishes a junctional complex and apical membrane specializations contacting the lumen of the gut. Finally, we show that the Drosophila homolog of ngn3, a bHLH gene that defines the entero-endocrine lineage in mammals, is expressed and required for the differentiation of this cell type in the fly gut.

Macrophage type 2 differentiation in a patient with laryngeal squamous cell carcinoma and metastatic prostate adenocarcinoma to the cervical lymph nodes

BackgroundThe tumor microenvironment often polarizes infiltrating macrophages towards a type 2, or M2 phenotype, that is characterized by expression of various cysteine-rich, scavenger receptors, including CD163. The primary function of M2 macrophages is to facilitate wound healing. As such, they are capable of providing metabolic support to a growing tumor, neovascularization, as well as protection from cytotoxic T cells. The tumor microenvironment contains a milieu of secreted factors and vesicles, which in certain circumstances can gain access to lymphatic vessels that drain to local lymph nodes.Case presentationWe report a 59-year-old male with recurrent T4 squamous cell carcinoma (SCC) of the larynx with synchronous prostate adenocarcinoma confined to the prostate and regional pelvic lymph nodes, without metastatic disease. The patient underwent salvage total laryngectomy and bilateral neck dissection with final pathology revealing a recurrent moderately differentiated SCC involving the larynx as well as prostate cancer in draining level 4 cervical lymph nodes bilaterally. CD163 staining was performed on the primary tumor, a negative draining lymph node, and a level four lymph node with a focus of metastatic prostate cancer and compared to benign controls. The negative draining lymph node demonstrated a large CD163 population of cells as did the interface of the focus of prostate cancer and surrounding lymph node. CD163 levels were markedly increased in this patient compared to benign lymph node controls. The macrophage differentiation at the primary tumor in the larynx was strongly CD163 positive supporting an immune permissive environment for tumor growth and metastasis.ConclusionWe describe a unique case of solitary metastatic prostate cancer to cervical lymph nodes in the setting of a laryngeal cancer. These observations suggest that SCC-derived factors drive a tumor-supportive environment in draining lymph nodes dominated by an overwhelming number of CD163+, M2 macrophages. Lymph nodes that are ‘primed’ by SCC differentiation to M2 phenotype may be at higher risk of harboring metastases.

Expression pattern of CD11c on lung immune cells after disseminated murine cytomegalovirus infection

BackgroundCytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection.MethodsMouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells.ResultsThis assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection.ConclusionsThe findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV.

MALDI MS Guided Liquid Microjunction Extraction for Capillary Electrophoresis-Electrospray Ionization MS Analysis of Single Pancreatic Islet Cells.

The ability to characterize chemical heterogeneity in biological structures is essential to understanding cellular-level function in both healthy and diseased states, but these variations remain difficult to assess using a single analytical technique. While mass spectrometry (MS) provides sufficient sensitivity to measure many analytes from volume-limited samples, each type of mass spectrometric analysis uncovers only a portion of the complete chemical profile of a single cell. Matrix-assisted laser desorption/ionization (MALDI) MS and capillary electrophoresis electrospray ionization (CE–ESI)-MS are complementary analytical platforms frequently utilized for single-cell analysis. Optically guided MALDI MS provides a high-throughput assessment of lipid and peptide content for large populations of cells, but is typically nonquantitative and fails to detect many low-mass metabolites because of MALDI matrix interferences. CE–ESI-MS allows quantitative measurements of cellular metabolites and increased analyte coverage, but has lower throughput because the electrophoretic separation is relatively slow. In this work, the figures of merit for each technique are combined via an off-line method that interfaces the two MS systems with a custom liquid microjunction surface sampling probe. The probe is mounted on an xyz translational stage, providing 90.6 ± 0.6% analyte removal efficiency with a spatial targeting accuracy of 42.8 ± 2.3 μm. The analyte extraction footprint is an elliptical area with a major diameter of 422 ± 21 μm and minor diameter of 335 ± 27 μm. To validate the approach, single rat pancreatic islet cells were rapidly analyzed with optically guided MALDI MS to classify each cell into established cell types by their peptide content. After MALDI MS analysis, a majority of the analyte remains for follow-up measurements to extend the overall chemical coverage. Optically guided MALDI MS was used to identify individual pancreatic islet α and β cells, which were then targeted for liquid microjunction extraction. Extracts from single α and β cells were analyzed with CE–ESI-MS to obtain qualitative information on metabolites, including amino acids. Matching the molecular masses and relative migration times of the extracted analytes and related standards allowed identification of several amino acids. Interestingly, dopamine was consistently detected in both cell types. The results demonstrate the successful interface of optical microscopy-guided MALDI MS and CE–ESI-MS for sequential chemical profiling of individual, mammalian cells.

Abstract 3789: Simultaneous visual assessment of RNA and protein expression in circulating tumor cells using the AccuCyte-Cytefinder system

Abstract Background. Circulating tumor cells (CTCs) provide real-time information regarding patient tumor phenotype, including RNA expression. This information may be valuable in guiding care of cancer patients. Simultaneous RNA and protein assessment has been reported previously in large populations of cells. Here, we describe methods to simultaneously evaluate RNA and protein expression on rare single CTCs using the AccuCyte – CyteFinder system (RareCyte). Methods. To establish RNA detection in model CTCs (mCTCs), we used SkBr3 cells spiked into blood and the isolated buffy coat was processed by AccuCyte onto microscope slides. RNAscope® in-situ hybridization (ISH, Advanced Cell Diagnostics) was performed to detect Her2, UBC (positive control) and dapB (negative control) expression in mCTCs. A process to simultaneously stain for RNA and protein was then developed to allow identification of mCTCs by protein expression of cytokeratin (CK) and EpCAM and measurement of gene expression with RNAscope. Various blood collection tubes were tested to measure gene expression, protein expression, and cell recovery up to 48 hours after blood draw. Image analysis software was developed to automatically analyze and count RNA dot number from 40x z-stack images of mCTCs. Finally, clinical samples were stained with the combined RNA/protein assay. Results. Using RNAscope, Her2 and UBC expression could be detected in all SkBr3 cells and was negative in surrounding white blood cells (WBC). Using blood collected into EDTA tubes and processed immediately after spike-in, there were an average of 29 Her2 mRNA dots and 20 UBC dots/cell; negative control dapB gave 1 dot/cell. Using CK and EpCAM expression for mCTCs identification, recovery of mCTCs was over 95%. The RNA/protein assay was compared using blood collected and stored in EDTA, CellSave®, Cell-Free DNA®, Cell-Free RNA®, Cyto-Chex®, and RareCyte BCT tubes. At 48 hours, Cell-Free RNA tubes had the highest number of RNA dots/cell – about half as many dots/cell compared to fresh EDTA samples. Clinical samples were successfully stained with the RNA/protein assay. Conclusions. We have developed a protocol that identifies rare mCTCs using protein staining, and measures RNA expression by RNAscope ISH. This assay may be a useful clinical tool for the real-time investigation of CTC gene expression in cancer patients. Citation Format: Arturo B. Ramirez, Sarah B. Crist, Tyler Yeats, Jeffrey L. Werbin, Jackie L. Stilwell, Eric P. Kaldjian. Simultaneous visual assessment of RNA and protein expression in circulating tumor cells using the AccuCyte-Cytefinder system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2017-3789

Saturation in Phosphene Size with Increasing Current Levels Delivered to Human Visual Cortex.

Electrically stimulating early visual cortex results in a visual percept known as a phosphene. Although phosphenes can be evoked by a wide range of electrode sizes and current amplitudes, they are invariably described as small. To better understand this observation, we electrically stimulated 93 electrodes implanted in the visual cortex of 13 human subjects who reported phosphene size while stimulation current was varied. Phosphene size increased as the stimulation current was initially raised above threshold, but then rapidly reached saturation. Phosphene size also depended on the location of the stimulated site, with size increasing with distance from the foveal representation. We developed a model relating phosphene size to the amount of activated cortex and its location within the retinotopic map. First, a sigmoidal curve was used to predict the amount of activated cortex at a given current. Second, the amount of active cortex was converted to degrees of visual angle by multiplying by the inverse cortical magnification factor for that retinotopic location. This simple model accurately predicted phosphene size for a broad range of stimulation currents and cortical locations. The unexpected saturation in phosphene sizes suggests that the functional architecture of cerebral cortex may impose fundamental restrictions on the spread of artificially evoked activity and this may be an important consideration in the design of cortical prosthetic devices.SIGNIFICANCE STATEMENT Understanding the neural basis for phosphenes, the visual percepts created by electrical stimulation of visual cortex, is fundamental to the development of a visual cortical prosthetic. Our experiments in human subjects implanted with electrodes over visual cortex show that it is the activity of a large population of cells spread out across several millimeters of tissue that supports the perception of a phosphene. In addition, we describe an important feature of the production of phosphenes by electrical stimulation: phosphene size saturates at a relatively low current level. This finding implies that, with current methods, visual prosthetics will have a limited dynamic range available to control the production of spatial forms and that more advanced stimulation methods may be required.

Extraocular Muscle Repair and Regeneration.

The goal of this review is to summarize the unique regenerative milieu within mature mammalian extraocular muscles (EOMs). This will aid in understanding disease propensity for and sparing of EOMs in skeletal muscle diseases as well as the recalcitrance of the EOM to injury. The EOMs continually remodel throughout life and contain an extremely enriched number of myogenic precursor cells that differ in number and functional characteristics from those in limb skeletal muscle. The EOMs also contain a large population of Pitx2-positive myogenic precursor cells that provide the EOMs with many of their unusual biological characteristics, such as myofiber remodeling and skeletal muscle disease sparing. This environment provides for rapid and efficient remodeling and regeneration after various types of injury. In addition, the EOMs show a remarkable ability to respond to perturbations of single muscles with coordinated changes in the other EOMs that move in the same plane. These data will inform Ophthalmologists as they work toward developing new treatments for eye movement disorders, new approaches for repair after nerve or direct EOMs injury, as well as suggest potential explanations for the unusual disease propensity and disease sparing characteristics of human EOM.