Ultrastructural visualization of the Mesenchymal-to-Epithelial Transition during reprogramming of human fibroblasts to induced pluripotent stem cells

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus, it has been demonstrated, that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells, with a concomitant repression of key mesenchymal genes. However, a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here, we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming, as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6–12 days after electroporation, in synchrony with the upregulation of early pluripotency markers, and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation

Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia

AbstractNeural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1–4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases

Coccidiose hos kvæg: En oversigt over coccidiearter, patogenese, epidemiologi og forebyggelse specielt i økologiske besætninger

Coccidiose hos kalve forårsages af arter af de encellede parasitter, Eimeria, der findes vidt udbredt i alle verdensdele, og som giver anledning til diarré og utrivelighed hos især unge og modtagelige dyr. Hos kvæg findes 12 forskellige Eimeria arter, der adskiller sig fra hinanden med hensyn til morfologi, livscyklus og patogenitet. Infektion med Eimeria kan diagnosticeres ved forskellige la-boratoriemetoder, hvorved bl.a. antallet og arten af coccidier forsøges fastslået. Således skabes grundlag for bestemmelse af hvilke forebyggende tiltag, der eventuelt bør iværksættes. Ved under-søgelse af 258 kalve fra 25 danske økologiske malkekvægsbesætninger fandt vi Eimeria-arter i alle (100%) besætninger og i 88% af kalvene. De fleste kalve udskilte meget lavgradige mængder af coccidier i gødningen, medens nogle havde særdeles høje coccidieudskillelser. Coccidiose er en multifaktoriel sygdom, og sværhedsgraderne af sygdommen varierer derfor fra subkliniske tilfælde til nogle med løs, pastøs fæces af en enkelt dags varighed eller profus diarré med dysenteri, tenesmus og evt. mors. De kliniske symptomer tillægges traditionelt tilstedeværelsen af coccidiearterne Eimeria zuernii og E. bovis. Kalvens modtagelighed for coccidiose afhænger bl.a. af råmælksforsyning og immunitet, ernæringsstatus, stress, og evt. forekomst af andre sygdomme (f.eks. samtidige helminth- eller virusinfektioner). Endelig har også miljøet betydning for udvikling af coccidiose, herunder opstaldningstæthed, klima og hygiejne. Total udryddelse af coccidier er ikke muligt pga. parasittens ubikvitære forekomst og resistens i miljøet, men kalven kan styrkes ved sikring af tilstrækkelige råmælksmængder og ved opretholdelse af høj hygiejne i kælvningsboksen, samt efterfølgende opstaldningsforhold. Aktiv immunisering (vaccination) har endnu ikke vist sig effektiv i større målestok. Der foreligger ikke yderligere retningslinier for forebyggelse i perspektiv af de økologiske regler. Kalve, der har klinisk coccidiose, bør isoleres fra de øvrige kalve og gives væsketerapi for at opretholde væskebalancen. Den bedste behandling er imidlertid forebyggelse - med fokus på belægningsgrad og høj hygiejne inklusive rigeligt med tør strøelse

Maximal accelerations for twelve weeks elicit improvement in a single out of a collection of cycling performance indicators in trained cyclists

INTRODUCTION: Cycling is a time-consuming sport. Cyclists, as many other athletes, therefore, focus on training effectively. The hypothesis was tested that twelve weeks of supplementary maximal acceleration training caused more favourable changes in cycling performance indicators as compared to changes measured in comparable control cyclists. METHODS: Trained cyclists (n = 24) participated. A control group and a group performing maximal acceleration training, as a supplement to their usual training, were formed. The maximal acceleration training consisted of series of ten repetitions of outdoor brief maximal accelerations, which were initiated from low speed and performed in a large gear ratio. The cyclists in the control group performed their usual training. Performance indicators, in form of peak power output in a 7-s maximal isokinetic sprint test, maximal aerobic power output in a graded test, and submaximal power output at a predetermined blood lactate concentration of 2.5 mmol L(−1) in a graded test were measured before and after the intervention. RESULTS: Peak power output in the sprint test was increased (4.1% from before to after the intervention) to a larger extent (p = 0.045) in the cyclists who had performed the maximal acceleration training than in the control cyclists (−2.8%). Changes in maximal aerobic power output and in submaximal power output at a blood lactate concentration of 2.5 mmol L(−1) were not significantly different between the groups (p > 0.351). DISCUSSION: The results indicated that the applied supplementary maximal acceleration training caused modest favourable changes of performance indicators, as compared to the changes measured in a group of comparable control cyclists

Ultrastructural myocardial changes in seven cats with spontaneous hypertrophic cardiomyopathy

AbstractObjectivesHypertrophic cardiomyopathy (HCM) is the most common heart disease in cats and shares clinical and pathological characteristics with human HCM. Little is known about the pathogenic mechanisms underlying development of spontaneous feline HCM.AnimalsThe study population consisted of seven cats diagnosed with HCM and eight age-matched cats with no evidence of cardiac disease.MethodsFresh myocardial biopsies taken from the middle of the left ventricular posterior free wall were obtained and examined with transmission electron microscopy.ResultsElectron microscopic examination showed ultrastructural aberrations of the myocardial cytoarchitecture and of the interstitium in the seven cats with HCM. In the most severely affected cats the myofibrils were disorganized and subsarcolemmal mitochondria were depleted. In control cats, contraction band artifacts were commonly seen.ConclusionsIn this preliminary study we show that ultrastructural changes of the myocardium in seven cats with HCM involve the cytoskeleton and mitochondria. We suggest that our findings are important for future research aiming at elucidating the pathogenic mechanisms underlying the phenotypic expression of feline HCM.The results of this study prompt for a larger scale study, including quantitative measurements of mitochondrial distribution and cytoskeletal derangements in feline HCM

Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry

This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electrical impedance spectroscopy in order to perform a theoretical study to clarify our results. This study focuses on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy