Physiological Heterogeneity Research Articles

Characterization of extracellular polymeric substances in the biofilms of typical bacteria by the sulfur K-edge XANES spectroscopy

A combined approach of physicochemical extraction and sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was applied to characterize the extracellular polymeric substances (EPS) of typical bacterial biofilms in this study. Physicochemical analysis showed variation of the contents of DNA, polysaccharide and protein in different fractions of EPS in different mediums. The sulfur K-edge XANES analysis yielded a variety of spectra. Spectral fitting of the XANES spectra utilizing a large set of model compounds showed that there was more reduced sulfur in both LB-EPS (loosely bound EPS) and TB-EPS (tightly bound EPS) of all the biofilms in LB medium than in R2A medium. More oxidized sulfur was identified in LB-EPS than that in TB-EPS, suggesting different niches and physiological heterogeneity in the biofilms. Our results suggested that the sulfur K-edge XANES can be a useful tool to analyze the sulfur speciation in EPS of biofilms.

The Application of Functional Imaging Techniques to Personalise Chemoradiotherapy in Upper Gastrointestinal Malignancies

Functional imaging gives information about physiological heterogeneity in tumours. The utility of functional imaging tests in providing predictive and prognostic information after chemoradiotherapy for both oesophageal cancer and pancreatic cancer will be reviewed. The benefit of incorporating functional imaging into radiotherapy planning is also evaluated. In cancers of the upper gastrointestinal tract, the vast majority of functional imaging studies have used 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). Few studies in locally advanced pancreatic cancer have investigated the utility of functional imaging in risk-stratifying patients or aiding target volume definition. Certain themes from the oesophageal data emerge, including the need for a multiparametric assessment of functional images and the added value of response assessment rather than relying on single time point measures. The sensitivity and specificity of FDG-PET to predict treatment response and survival are not currently high enough to inform treatment decisions. This suggests that a multimodal, multiparametric approach may be required. FDG-PET improves target volume definition in oesophageal cancer by improving the accuracy of tumour length definition and by improving the nodal staging of patients. The ideal functional imaging test would accurately identify patients who are unlikely to achieve a pathological complete response after chemoradiotherapy and would aid the delineation of a biological target volume that could be used for treatment intensification. The current limitations of published studies prevent integrating imaging-derived parameters into decision making on an individual patient basis. These limitations should inform future trial design in oesophageal and pancreatic cancers.

Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: an innovation in plant cell preservation.

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80°C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0°C and 25°C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.

Oxygen limitation within a bacterial aggregate.

ABSTRACTCells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >108 cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤105 bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D “microtraps” that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (1012 cells ml−1) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations.

Ventricular Assist Devices in Children

Pediatric mechanical circulatory support (MCS) has advanced tremendously over the last decade, with momentum building in the development and implantation of durable ventricular assist devices (VADs) in smaller children with increasingly complex anatomy. Despite these advancements, VAD support for children continues to lag behind support options for adults. The most obvious disparity is the limited number of devices suitable for children for both short- and long-term support. Thus, there has been burgeoning interest in developing devices specifically tailored to suit the unique needs of children. From a design perspective, pediatric VADs differ significantly from adult devices: they are required to support a wide range of patient sizes (from newborn to adolescence), to allow for the increased circulatory demand commensurate with growth, and to accommodate the anatomic and physiological heterogeneity of congenital heart disease. Beyond the challenges of designing and developing pediatric VADs, there are multiple barriers to the clinical evaluation of these devices because of the small number of children who require this level of support. Therefore, it is considered infeasible to test device safety and effectiveness through large randomized, controlled trials like the Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure (REMATCH),1 a large adult study. Additionally, industry is disincentivized to invest the necessary resources in the research and development of pediatric-specific medical devices with applicability to such a narrow market population.2 In the United States, the Food and Drug Administration (FDA) is tasked with the responsibility of ensuring that medical devices marketed in the United States have met the requirements establishing the safety and effectiveness for use in humans to treat a specific condition. Many pediatric-specific medical devices, including VADs, fall under the category of a humanitarian use device.3 Humanitarian use devices are devices that are used to treat or diagnose …

Strategies for using the sheep ovarian cortex as a model in reproductive medicine.

ObjectiveTo evaluate and compare the distribution and density of primordial follicles within a whole sheep ovary and to gain insight into how to overcome the impact of natural follicular heterogeneity on the experimental results.DesignHistological study.SettingAcademic research center.AnimalsFive- to nine-month-old ewes.InterventionsFreshly sampled whole sheep ovaries were collected and prepared for histological analysis.Main Outcome Measure(s)The follicular densities and distributions were determined for hematoxylin and eosin sections. A mathematical model was derived based on the follicle counts and Monte-Carlo simulations.ResultsHeterogeneous distributions and densities of primordial follicles were identified 1) for distinct areas of the same ovarian cortex, 2) between the ovaries of the same animal and 3) across different ewes. A mathematical model based on the analysis of 37,153 primordial follicles from 8 different ovaries facilitated the estimation of the number of cortical biopsies and sections that had to be analyzed to overcome such heterogeneity.ConclusionThe influence of physiological follicular heterogeneity on experimental and clinical results can be overcome when a definite number of cortical pieces and sections are taken into consideration.

Physical and Physiological Heterogeneity within the Grape Bunch: Impact on Mechanical Properties during Maturation

Mechanical properties are emerging as complementary criteria to evaluate grape berry quality for winemaking. As texture is evaluated on a berry per berry basis, it is important to assess the impact of the within grape heterogeneity on mechanical properties. Evolution of textural maturity of Cabernet franc was assayed throughout ripening using double compression 20% tests. Berries were sampled from whole grape bunches rather than randomly picked in the vineyard. Results showed coherent and significant evolution of double compression parameters: F1, Grad0, Grad1, W1, and gumminess significantly decreased whereas cohesiveness significantly increased during maturation. Variations in mechanical parameters were related with major physiological and physical traits. Mechanical parameters differed significantly according to berry position. Among these parameters, Grad1 was highly correlated with berry density and Brix (r = −0.91 for both), and W1 was highly correlated with water content (r = 0.87). Within-bunch heterogeneity was illustrated by different distributions of physical, physiological, and mechanical parameters. A negative gradient was shown from the top to the bottom of bunches for dry weight, density, Brix, cohesiveness, and gumminess. Berries at the distal part of the bunch were more advanced in maturity and were softer than berries at the top. Results are discussed with regard to the sampling strategy in the assessment of grape berry quality based on mechanical properties.

Physiological heterogeneity in Lactobacillus casei fermentations on residual yoghurt whey

Lactobacillus casei is a well-known lactic acid-producer with substantial industrial interest. Currently, inexpensive lactic acid substrates such as residual yoghurt whey are being increasingly employed as revalorization strategies for such polluting food industry wastes. However, the influence of different bioprocessing conditions on the cellular functionality and physiological status of L. casei at single cell level has barely been evaluated to date. In the present study, monitoring the different physiological states of L. casei through multiparametric flow cytometry during lactic acid production from residual yoghurt whey showed that the majority of L. casei cells remained in healthy, metabolically active state (∼70%) under uncontrolled-pH conditions (pH <3.6), whereas a progressive increase in population heterogeneity was determined (increasing the damaged and dead subpopulations) with higher production (41.5g/L lactate titer) and sugar consumption rates when a pH-controlled strategy at 6.5 was adopted. A segregated kinetic model was additionally developed to better describe the physiological behaviour of microbial heterogeneity, gaining deeper knowledge on the lactic acid-producing ability of each subpopulation under pH-controlled conditions in the mixed sugar co-fermentation. This study provides further understanding on the role of physiological heterogeneity in lactobacilli populations useful to enhance bioprocess performance and thus achieve efficient lactic acid production.

Gut Commensal Bacteria and Regional Wnt Gene Expression in the Proximal Versus Distal Colon

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length.These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/β-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria.

Morpho‐physiological criteria divide dentate gyrus interneurons into classes

GABAergic inhibitory interneurons control fundamental aspects of neuronal network function. Their functional roles are assumed to be defined by the identity of their input synapses, the architecture of their dendritic tree, the passive and active membrane properties and finally the nature of their postsynaptic targets. Indeed, interneurons display a high degree of morphological and physiological heterogeneity. However, whether their morphological and physiological characteristics are correlated and whether interneuron diversity can be described by a continuum of GABAergic cell types or by distinct classes has remained unclear. Here we perform a detailed morphological and physiological characterization of GABAergic cells in the dentate gyrus, the input region of the hippocampus. To achieve an unbiased and efficient sampling and classification we used knock-in mice expressing the enhanced green fluorescent protein (eGFP) in glutamate decarboxylase 67 (GAD67)-positive neurons and performed cluster analysis. We identified five interneuron classes, each of them characterized by a distinct set of anatomical and physiological parameters. Cross-correlation analysis further revealed a direct relation between morphological and physiological properties indicating that dentate gyrus interneurons fall into functionally distinct classes which may differentially control neuronal network activity. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.

Regional Alveolar Partial Pressure of Oxygen Measurement with Parallel Accelerated Hyperpolarized Gas MRI

Alveolar oxygen tension (Pao2) is sensitive to the interplay between local ventilation, perfusion, and alveolar-capillary membrane permeability, and thus reflects physiologic heterogeneity of healthy and diseased lung function. Several hyperpolarized helium ((3)He) magnetic resonance imaging (MRI)-based Pao2 mapping techniques have been reported, and considerable effort has gone toward reducing Pao2 measurement error. We present a new Pao2 imaging scheme, using parallel accelerated MRI, which significantly reduces measurement error. The proposed Pao2 mapping scheme was computer-simulated and was tested on both phantoms and five human subjects. Where possible, correspondence between actual local oxygen concentration and derived values was assessed for both bias (deviation from the true mean) and imaging artifact (deviation from the true spatial distribution). Phantom experiments demonstrated a significantly reduced coefficient of variation using the accelerated scheme. Simulation results support this observation and predict that correspondence between the true spatial distribution and the derived map is always superior using the accelerated scheme, although the improvement becomes less significant as the signal-to-noise ratio increases. Paired measurements in the human subjects, comparing accelerated and fully sampled schemes, show a reduced Pao2 distribution width for 41 of 46 slices. In contrast to proton MRI, acceleration of hyperpolarized imaging has no signal-to-noise penalty; its use in Pao2 measurement is therefore always beneficial. Comparison of multiple schemes shows that the benefit arises from a longer time-base during which oxygen-induced depolarization modifies the signal strength. Demonstration of the accelerated technique in human studies shows the feasibility of the method and suggests that measurement error is reduced here as well, particularly at low signal-to-noise levels.

Electrophysiological Heterogeneity of Fast-Spiking Interneurons: Chandelier versus Basket Cells

In the prefrontal cortex, parvalbumin-positive inhibitory neurons play a prominent role in the neural circuitry that subserves working memory, and alterations in these neurons contribute to the pathophysiology of schizophrenia. Two morphologically distinct classes of parvalbumin neurons that target the perisomatic region of pyramidal neurons, chandelier cells (ChCs) and basket cells (BCs), are generally thought to have the same “fast-spiking” phenotype, which is characterized by a short action potential and high frequency firing without adaptation. However, findings from studies in different species suggest that certain electrophysiological membrane properties might differ between these two cell classes. In this study, we assessed the physiological heterogeneity of fast-spiking interneurons as a function of two factors: species (macaque monkey vs. rat) and morphology (chandelier vs. basket). We showed previously that electrophysiological membrane properties of BCs differ between these two species. Here, for the first time, we report differences in ChCs membrane properties between monkey and rat. We also found that a number of membrane properties differentiate ChCs from BCs. Some of these differences were species-independent (e.g., fast and medium afterhyperpolarization, firing frequency, and depolarizing sag), whereas the differences in the first spike latency between ChCs and BCs were species-specific. Our findings indicate that different combinations of electrophysiological membrane properties distinguish ChCs from BCs in rodents and primates. Such electrophysiological differences between ChCs and BCs likely contribute to their distinctive roles in cortical circuitry in each species.

Stress response assessment of Lactobacillus sakei strains selected as potential autochthonous starter cultures by flow cytometry and nucleic acid double-staining analyses

The aim of this study was to apply the flow cytometry to Lactobacillus sakei strains, selected as potential autochthonous starters, to investigate dynamics and physiological heterogeneity of microbial behaviour under different stress conditions. A simultaneous nucleic acid double-staining assay was applied to discriminate cell populations in different physiological states after exposure to heat (50 and 55°C) and acid (pH 2·5 and 3·0) stresses. Alive cells with intact membranes, damaged cells still alive but with injured membranes, so with even a recovery ability, and dead cells with a permanent membrane damage were differentiated with a significant increase in damaged cells after stronger stress treatments. The existence and characteristics of subpopulations displaying heterogeneity in particular conditions are highly relevant, because specific subpopulations may show improved survival, changes and dynamics under stress conditions. This assay has potential for physiological research on lactic acid bacteria and for application in the food industry. The assessment of intermediate physiological states in Lb.sakei strains with recovery possibility could be an important criterion for application of potential starter cultures. Application of flow cytometry and characterization of sorted subpopulations may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations.

Distinctive profile of IsomiR expression and novel microRNAs in rat heart left ventricle.

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.

The heterogeneity and spatial patterning of structure and physiology across the leaf surface in giant leaves of Alocasia macrorrhiza.

Leaf physiology determines the carbon acquisition of the whole plant, but there can be considerable variation in physiology and carbon acquisition within individual leaves. Alocasia macrorrhiza (L.) Schott is an herbaceous species that can develop very large leaves of up to 1 m in length. However, little is known about the hydraulic and photosynthetic design of such giant leaves. Based on previous studies of smaller leaves, and on the greater surface area for trait variation in large leaves, we hypothesized that A. macrorrhiza leaves would exhibit significant heterogeneity in structure and function. We found evidence of reduced hydraulic supply and demand in the outer leaf regions; leaf mass per area, chlorophyll concentration, and guard cell length decreased, as did stomatal conductance, net photosynthetic rate and quantum efficiency of photosystem II. This heterogeneity in physiology was opposite to that expected from a thinner boundary layer at the leaf edge, which would have led to greater rates of gas exchange. Leaf temperature was 8.8°C higher in the outer than in the central region in the afternoon, consistent with reduced stomatal conductance and transpiration caused by a hydraulic limitation to the outer lamina. The reduced stomatal conductance in the outer regions would explain the observed homogeneous distribution of leaf water potential across the leaf surface. These findings indicate substantial heterogeneity in gas exchange across the leaf surface in large leaves, greater than that reported for smaller-leafed species, though the observed structural differences across the lamina were within the range reported for smaller-leafed species. Future work will determine whether the challenge of transporting water to the outer regions can limit leaf size for plants experiencing drought, and whether the heterogeneity of function across the leaf surface represents a particular disadvantage for large simple leaves that might explain their global rarity, even in resource-rich environments.

Maternal serum leptin during pregnancy and infant birth weight: The influence of maternal overweight and obesity

Few studies have examined whether the distinct metabolic patterns found in obese and non-obese pregnant women may have different effects on the growing fetus. Our objective was to estimate the influence of longitudinal variation in maternal serum leptin levels on variation in infant birth weight in overweight/obese versus normal weight women. In a prospective cohort of 286 gravidas, we measured maternal weight and serum leptin levels at 6–10,10–14,16–20, 22–26, and 32–36 weeks gestation. Effects of leptin levels on infant birth weight adjusted for gestational age at delivery (aBW) were analyzed using a linear regression model that accounted for the relationship of time-varying predictors to the log transformed leptin concentrations. Overweight/obese and normal weight gravidas exhibit different relationships of aBW to maternal serum leptin and its rate of change across pregnancy. For normal weight women, aBW is not associated with either the magnitude of the logarithm of the leptin concentration nor with its rate of change in either the first or second half of pregnancy. Conversely, for overweight/obese women, we find that an increase in the rate of change in maternal serum leptin in the second half of pregnancy is significantly associated with a decrease in aBW. We find that this effect is distinct from that of maternal weight. Differences in the effect of maternal serum leptin on fetal growth between overweight/obese and normal weight women suggest metabolic and physiologic heterogeneity between these groups. Such differences may be involved in the long-term physiologic effects of the obese intrauterine environment on the health of the offspring.

Reply to “Precedence for the Structural Role of Flagella in Biofilms”

In the Serra et al. paper (1), we addressed the question of spatial physiological heterogeneity in Escherichia coli macrocolonies that were grown for up to 7 days on agar plates. These are architectonically structured biofilms that develop at a wet medium/air interface. In essence, we observed a two-layer architecture (with a transition zone of changing physiology in between). The bottom layer, which is close to the nutrient-providing agar and extends to the rims of the colonies, exhibits the visual hallmarks of a vegetative growth zone with rod-shaped dividing cells that produce flagella, which get entangled by rotation and thereby generate a filamentous mesh that contributes to cohesion of the colony and overall macrostructure. In contrast, the surface layer consists of ovoid starving cells that are surrounded by a thick network of σS-controlled amyloid curli fibers that are essential to produce complex macrocolony structures. One of the outcomes of our study is the proposal of a novel architectural role of flagella in macrocolony biofilms that is not related to motility. In the Discussion, we related our findings to earlier observations that flagella in general are involved in biofilm formation (2, 3). However, we refrain from extending our proposal to submerged biofilms in flow cell devices for several reasons. Not only does the latter type of model biofilm have a “mushroom”-like architecture different from macrocolonies, …

Regional Wnt signatures in the colon and the influence of commensal bacteria

Wnt signaling is a key regulator of intestinal epithelial homeostasis. Since the large intestine exhibits considerable morphological and functional heterogeneity along its length, we investigated whether this can be explained by local differences in Wnt signaling. Furthermore, we examined whether divergent Wnt signaling may be shaped by commensal bacteria. Using a Wnt PCR array, expression of 84 genes related to Wnt‐mediated signal transduction was analyzed. A differential signature of Wnt related genes in the proximal vs. distal murine colon was identified. Specifically, we observed that Wnt agonists Wnt5a, Wnt8b, and Wnt11, Wnt receptors Fzd3 and Lrp6, and a Wnt inhibitory factor Wif1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal vs. distal colon. Given the importance of luminal bacteria in controlling epithelial homeostasis, Wnt gene expression in colonic segments of mice raised in germ‐free conditions were analyzed. Altered expression of Wnt genes was identified in mice raised in germ‐free vs. specific pathogen free conditions. These results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by differential expression of Wnt signaling components and influenced by colonization with bacteria.

Next‐generation sequencing analysis of rat heart left ventricle: novel microRNAs, novel isomiRs and transmural distribution of microRNA expression

MicroRNAs (miRNAs) have a significant role in regulating gene expression in heart disease. They can exist as isoforms (isomiRs) that are potentially (patho)physiologically important. The heterogeneity of cardiomyocyte physiology across the left ventricular wall has an important influence on heart (patho)physiology, and may well be affected by transmural miRNA expression gradients. With the aims of determining rat heart isomiR profiles and left ventricular transmural miRNA expression we investigated epi/midmyo/endocardial tissue samples using Illumina next‐generation sequencing. We detected significant quantities of 146 known rat miRNAs and 68 novel orthologs of known miRNAs. Many novel isomiRs were detected at a higher frequency than the corresponding mature sequence, and have predicted targets different from those of the corresponding canonical miRNA. We identified a novel rat miR‐1 analogue from a second miR‐1 gene and a novel rat miRNA similar to miR‐676. We also cloned and sequenced the rat miR‐486 gene. Several important cardiac intracellular signalling pathways are predicted targets of the most highly detected miRNAs. Most miRNAs are not expressed in a gradient across the ventricular wall; the exceptions include miR‐10b, miR‐21, miR‐99b and miR‐486. These findings suggest new mechanisms of cardiac gene regulation in health and disease. Funded by British Heart Foundation PG110/039/28365.

Microfluidic Single Mammalian Cell Lysis in Picolitre Droplets

One of the essential issues in cell biology is to analyze intracellular proteins that play an important role in signal transduction and processes. Standard assays for intracellular proteins are based on ensemble measurements which rule out the differences of intracellular protein expression in individual cells. Especially in cancer biology, only a few tumour cells turn into circulating tumour cells causing metastasis. In such cases, understanding the heterogeneity in cellular physiology would be necessary. To screen for intracellular protein expression among individual cells effectively, a reliable method to lyse individual cells in a high throughput manner is essential. Here, we demonstrate an integrated microfluidic platform combining a cell focusing channel, reagent injection inlet and a droplet generator for high throughput single cell lysis. Individual cells are encapsulated into the droplets with chemical reagents which result in disrupting the cell membrane integrity, causing cell lysis. High throughput mammalian cell lysis was performed thus facilitating intracellular protein analysis on a micro device