A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min

The molecular phylogeny of eph receptors and ephrin ligands

<p>Abstract</p> <p>Background</p> <p>The tissue distributions and functions of Eph receptors and their ephrin ligands have been well studied, however less is known about their evolutionary history. We have undertaken a phylogenetic analysis of Eph receptors and ephrins from a number of invertebrate and vertebrate species.</p> <p>Results</p> <p>Our findings indicate that Eph receptors form three major clades: one comprised of non-chordate and cephalochordate Eph receptors, a second comprised of urochordate Eph receptors, and a third comprised of vertebrate Eph receptors. Ephrins, on the other hand, fall into either a clade made up of the non-chordate and cephalochordate ephrins plus the urochordate and vertebrate ephrin-Bs or a clade made up of the urochordate and vertebrate ephrin-As.</p> <p>Conclusion</p> <p>We have concluded that Eph receptors and ephrins diverged into A and B-types at different points in their evolutionary history, such that primitive chordates likely possessed an ancestral ephrin-A and an ancestral ephrin-B, but only a single Eph receptor. Furthermore, ephrin-As appear to have arisen in the common ancestor of urochordates and vertebrates, whereas ephrin-Bs have a more ancient bilaterian origin. Ancestral ephrin-B-like ligands had transmembrane domains; as GPI anchors appear to have arisen or been lost at least 3 times.</p

Factors associated with disease evolution in Greek patients with inflammatory bowel disease

BACKGROUND: The majority of Crohn's disease patients with B1 phenotype at diagnosis (i.e. non-stricturing non-penetrating disease) will develop over time a stricturing or a penetrating pattern. Conflicting data exist on the rate of proximal disease extension in ulcerative colitis patients with proctitis or left-sided colitis at diagnosis. We aimed to study disease evolution in Crohn's disease B1 patients and ulcerative colitis patients with proctitis and left-sided colitis at diagnosis. METHODS: 116 Crohn's disease and 256 ulcerative colitis patients were followed-up for at least 5 years after diagnosis. Crohn's disease patients were classified according to the Vienna criteria. Data were analysed actuarially. RESULTS: B1 phenotype accounted for 68.9% of Crohn's disease patients at diagnosis. The cumulative probability of change in disease behaviour in B1 patients was 43.6% at 10 years after diagnosis. Active smoking (Hazard Ratio: 3.01) and non-colonic disease (non-L2) (Hazard Ratio: 3.01) were associated with behavioural change in B1 patients. Proctitis and left-sided colitis accounted for 24.2%, and 48.4% of ulcerative colitis patients at diagnosis. The 10 year cumulative probability of proximal disease extension in patients with proctitis and left-sided colitis was 36.8%, and 17.1%, respectively (p: 0.003). Among proctitis patients, proximal extension was more common in non-smokers (Hazard Ratio: 4.39). CONCLUSION: Classification of Crohn's disease patients in B1 phenotype should be considered as temporary. Smoking and non-colonic disease are risk factors for behavioural change in B1 Crohn's disease patients. Proximal extension is more common in ulcerative colitis patients with proctitis than in those with left-sided colitis. Among proctitis patients, proximal extension is more common in non-smokers

Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>Pax3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few Pax3 target genes have been identified. Identifying genes that lie genetically downstream of <it>Pax3 </it>is therefore an important endeavour in elucidating the myogenic gene regulatory network.</p> <p>Results</p> <p>We have undertaken a screen in the mouse embryo which employs a <it>Pax3^GFP</it>allele that permits isolation of Pax3 expressing cells by flow cytometry and a <it>Pax3^PAX3-FKHR</it>allele that encodes PAX3-FKHR in which the DNA binding domain of Pax3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the <it>Pax3 </it>mutant phenotype. Microarray comparisons were carried out between <it>Pax3^GFP/+</it>and <it>Pax3^{GFP/PAX3-FKHR}</it>preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between Pax3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential Pax3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function <it>Pax3 </it>mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from Pax3 positive neural crest cells which do not invade the limbs. Verification by whole mount <it>in situ </it>hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.</p> <p>Conclusions</p> <p>Pax3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate Pax3 targets. Myogenic determination genes, such as <it>Myf5 </it>are controlled positively, whereas the effect of <it>Pax3 </it>on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, <it>Pax7 </it>and also <it>Hdac5 </it>which is a potential repressor of <it>Foxc2</it>, are subject to positive control by <it>Pax3</it>.</p

Detection of Batrachochytrium dendrobatidis in amphibians imported into the UK for the pet trade

There is increasing evidence that the global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been facilitated by the international trade in amphibians. Bd was first detected in the UK in 2004, and has since been detected in multiple wild amphibian populations. Most amphibians imported into the UK for the pet trade from outside the European Union enter the country via Heathrow Animal Reception Centre (HARC), where Bd positive animals have been previously detected. Data on the volume, diversity and origin of imported amphibians were collected for 59 consignments arriving at HARC between November 2009 and June 2012, along with a surveillance study to investigate the prevalence of Bd in these animals. Forty three amphibian genera were recorded, originating from 12 countries. It was estimated that 5000 – 7000 amphibians are imported through HARC into the UK annually for the pet trade. Bd was detected in consignments from the USA and Tanzania, in six genera, resulting in an overall prevalence of 3.6%. This suggests that imported amphibians are a source of Bd within the international pet trade

DNA DAMAGE INDUCED BY EXERCISE IN MIDDLE GLUTEAL MUSCLE OF THOROUGHBREDS HORSES

ABSTRACT It is well known that two classical consequences of severe exercise are soreness and stiffness. Both consequences develop in the days following severe exercise. The objective of the present work was to establish the association between DNA damage, the energetic metabolism and the effects of the generation of oxygen free radicals in middle gluteal muscle of horses, measured after a severe exercise bout on a treadmill. Four thoroughbreds, ranging in aged from 3 to 4 years old were used for this study. The test consisted in warming-up, severe exercise, and cooling down. Venous blood samples were withdrawn via a catheter from the jugular vein before and at 5 min, 30 min, 45 min and 24 h post-exercise. Muscle biopsy samples were obtained from the middle gluteal muscle before and at 45 min and 24 h after exercise. All statistical analyses were performed using Student&apos;s t-tests for unpaired data. ATP concentration significantly decreased (p&lt; .05) after 45 min of severe exercise and subsequently increased by 24 h. Plasma CK activity increased significantly (p&lt; .05) after 45 min and 24 h of exercise. Both free MDA and protein-bound MDA concentrations significantly increased (p&lt; .001) after 45 min of exercise, and both of them returned almost to the pre-exercise values after 24 h of exercise. The percentage of TUNEL positive cells increased significantly (p&lt; .001) so as the plasma hypoxanthine and uric acid level during exercise. It was strongly suggested that the generation of free radicals was developed during exercise. The mechanism of exercise-induced myopathy could be the cell membrane damage by lipid peroxidation and DNA fragmentation in the working muscle cells by free radicals generation. RESUMEN Dos consecuencias clásicas del ejercicio severo son el dolor y la rigidez. Ambas consecuencias ocurren en los días siguientes al ejercicio. El objetivo de este trabajo fue establecer la asociación entre daño en el ADN, el metabolismo energético y la generación de radicales del oxígeno en el músculo gluteo medio de caballos sometidos a ejercicio severo. Se utilizaron 4 caballos de entre 3 y 4 años de edad, que fueron sometidos a ejercicio en cinta ergométrica. Se obtuvieron muestras de sangre por punción yugular antes, a los 5, 30, 45 minutos y 24 horas pos ejercicio. Se realizaron biopsias del músculo gluteo medio antes, a los 45 minutos y 24 horas pos ejercicio. Se realizaron múltiples determinaciones y mediciones y en todos los casos el análisis estadístico se realizó utilizando el test de t para muestras no apareadas. La concentración de ATP disminuyó significativamente a los 45 minutos de ejercicio severo (p&lt; 0, 05) y se recupero a las 24 hs. La actividad plasmática CK se incremento significativamente (p&lt; .05) a los 45 minutos y 24 h de realizado el ejercicio. Tanto el MDA libre como el fijado a proteínas aumentaron sus concentraciones significativamente (p&lt; .001) a 45 minutos, y ambos volvieron a los valores de pre ejercicio a las 24 horas. El porcentaje de células tunnel positive y los niveles plasmáticos de hipoxantina y ácido úrico se incrementaron signioficativamente durante el ejercicio (p&lt; .001). Los datos obtenidos sugieren la generación de radicales libres durante el ejercicio y la producción de daño en el ADN. El mecanismo por el cual el ejercicio induce miopátias puede deberse al daño de membranas celulares por peroxidación de lípidos y a la fregmentación del ADN, debida a la producción de radicales libres

Effect of zinc on DNA integrity of cumulus cells during oocyte in Vitro maturation ZINC AND DNA INTEGRITY OF CUMULUS CELLS

ABSTRACT Zinc is an essential trace element. It has been known that a non adequate level of zinc can alter not only gene expression but also a variety of cellular functions with severe consequences on animal health. Little is known about the role of zinc on the reproductive performance of bovines, especially in oocyte maturation. The objective of the present study was to evaluate the impact of different zinc concentrations added to culture medium, on the DNA integrity of bovine cumulus cells during in vitro maturation of oocytes. Cumulus-oocyte coplexes obtained from an abattoir were cultured in IVM mediums containing 30 (G1), 70 (G2), 110 (G3) and 150 (G4) µg/dl. At the end of IVM, cumulus cells were processed for Comet Assay. Result obtained with the comet assay shows a significant decrease in the percentage of cell with DNA migration from around 40% in G1 and G2 to 20% in G3 and G4 (p&lt; 0,05). The intensity of DNA damage (DDV) was higher in G1 and G2 and significant different with G3 and G4 (p&lt; 0,05). Taken into account, the relationship between in vitro maturation of cumulus cells oocytes complexes (COCs) and subsequent embryo development, it might be possible that different levels on Zinc concentration during IVM have consequences on early embryo development. Key Word: Zinc, cumulus cells, comet assay RESUMEN El zinc es un micronutriente cuya deficiencia afecta no solo la expresión génica, sino también una variedad de funciones celulares con consecuencias directas en la salud animal. Poco se conoce acerca del rol del zinc en la performance reproductiva de los bovinos, especialmente en relación con la maduración de los ovocitos. El objetivo del presente trabajo fue evaluar la integridad del ADN de en células del cúmulus de bovinos, cultivadas con diferentes concentraciones de zinc adicionadas a el medio de cultivo de maduración in Vitro de ovocitos. Para ello, los complejos cúmulos-ovocitos obtenidos en frigoríficos fueron cultivados en Medios para Maduración in Vitro, conteniendo 30 (G1), 70 (G2), 110 (G3) y 150 (G4) µg/dl de zinc. Al finalizar la maduración, las células del cúmulos fueron procesadas para realizar el ensayo cometa. Los resultados obtenidos con dicho ensayo mostraron una disminución significativa en el porcentaje de células con migración del ADN, de alrededor del 40% en G1 y G2 hasta alrededor de 20% en G3 y G4 (p&lt; 0,05). La intensidad del daño en el ADN fue significativamente mayor en G1 y G2 que en G3 y G4 (p&lt; 0,05). Considerando la estrecha relación existente entre los complejos ovocito-células del cúmulus y el subsecuente desarrollo embrionario, es posible inferir que diferentes niveles de Zinc durante la MIV pueden tener consecuencias en el desarrollo embrionario temprano. Palabras claves: zinc, células del cúmulus, ensayo cometa

A study of dynamic nanoscale corrosion initiation events using HS-AFM

Atomic force microscopes (AFMs) are capable of high-resolution mapping of structures and the measurement of mechanical properties on nanometre scales within gaseous, liquid and vacuum environments. The contact mode high-speed AFM (HS-AFM) developed at Bristol Nano Dynamics Ltd. operates at speeds that are orders of magnitude faster than conventional AFMs, and is capable of capturing multiple frames per second. This allows for direct observation of dynamic events in real-time, with nanometre lateral resolution and subatomic height resolution. HS-AFM is a valuable tool for the imaging of nanoscale corrosion initiation events, such as metastable pitting, grain boundary (GB) dissolution and short crack formation during stress corrosion cracking (SCC). Within this study HS-AFM was combined with SEM and FIB milling to produce a multifaceted picture of localised corrosion events occurring on thermally sensitised AISI 304 stainless steel in an aqueous solution of 1% sodium chloride (NaCl). HS-AFM measurements were performed in situ by imaging within a custom built liquid cell with parallel electrochemical control. The high resolution of the HS-AFM allowed for measurements to be performed at individual reaction sites, i.e. at specific GB carbide surfaces. Topographic maps of the sample surface allowed for accurate measurements of the dimensions of pits formed. Using these measurements it was possible to calculate, and subsequently model, the volumes of metal reacting with respect to time, and so the current densities and ionic fluxes at work. In this manner, the local electrochemistry at nanoscale reaction sites may be reconstructed