Engineered microenvironments for synergistic VEGF - integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications

Horizontal transfers of two types of puf operons among phototrophic members of the Roseobacter clade

The Roseobacter clade represents one of the most important bacterial groups in marine environments. While some of its members are heterotrophs, many Roseobacter clade members contain bacterial photosynthetic reaction centers. We investigated the phylogeny of pufL and pufM genes encoding the L and M subunits of reaction centers using available genomic data and our own cultured species. Interestingly, phylogeny of pufL and pufM genes largely deviated from 16S rRNA-based phylogeny. The sequences split into two clearly distinct clades. While most of the studied species contained pufL and pufM sequences related to those found in Roseobacter litoralis, some of the marine species contained sequences related to the freshwater Rhodobacter species. In addition, genomic data documents that Roseobacter-type centers contain cytochrome c subunits (pufC gene product), whereas Rhodobacter-type centers incorporate PufX proteins. This indicates that the two forms of the reaction centers are not only distinct phylogenetically, but also structurally. The large deviation of pufL and pufM phylogeny from 16S phylogeny indicates multiple horizontal transfers of the puf operon among members of the order Rhodobacterales

Artificial neural networks and computer vision in medicine and surgery

Úvod: Umělé neuronové sítě se stávají důležitou technologií při analýze dat a jejich vliv začíná prostupovat i do oblasti medicíny. Naše pracoviště se dlouhodobě věnuje experimentální chirurgii, na to navazuje náš zájem o pokrok v ostatních oblastech moderních technologií a tím i umělých neuronových sítí. V rámci aktuálního čísla chceme prozkoumat i tento aspekt technického pokroku. Hlavním cílem je kritické zhodnocení silných i slabých stránek technologie umělých neuronových sítí s ohledem na využití v klinické a experimentální chirurgii. Metody: V článku je věnována pozornost in-silico modelování a zejména pak možnostem neuronových sítí s ohledem na zpracování obrazových dat v medicíně. V textu je krátce shrnut historický vývoj hlubokého učení neuronových sítí a základní principy jejich fungování. Dále je představena taxonomie základních řešených úloh. Zmíněny jsou i možné problémy při učení i s možnostmi jejich řešení. Výsledky: Článek poukazuje na rozličné možnosti umělých neuronových sítí v biologických aplikacích. Na řadě biomedicínských aplikací umělých neuronových sítí popisuje rozdělení a princip základních úloh strojového učení a hlubokého učení - klasifikace, detekce a segmentace. Závěr: Aplikace metod umělých neuronových sítí mají v medicíně a chirurgii značný potenciál. Obcházejí potřebu zdlouhavého subjektivního nastavování parametrů znalostním inženýrem, neboť se učí přímo z dat. Při využití nevhodně vyváženého datasetu však může docházet k neočekávaným, avšak zpětně vysvětlitelným chybám. Řešení představuje vytvoření dostatečně bohatého datasetu pro učení a ověření funkceIntroduction: Artificial neural networks are becoming an essential technology in data analysis, and their influence is starting to permeate the field of medicine. Experimental surgery has been a long-term subject of study of our lab; this is naturally reflected in our interest in other areas of modern technologies including artificial neural networks and their advancements. In the current issue, we would like to explore this aspect of technical progress. The main goal is to critically evaluate the strengths and weaknesses of artificial neural network technology concerning its use in clinical and experimental surgery. Methods: The article is focused on in-silico modeling, particularly on the potential of neural networks in terms of image data processing in medicine. The text briefly summarizes the historical development of deep learning neural networks and their basic principles. Furthermore, basic taxonomy tasks are presented. Finally, potential learning problems and possible solutions are also mentioned. Results: The article points out various possible uses of artificial neural networks in biological applications. Several biomedical applications of artificial neural networks are used to describe the division and principles of the most common tasks of machine learning and deep learning such as classification, detection, and segmentation. Conclusion: The application of artificial neural network methods in medicine and surgery offers a considerable potential; by learning directly from the data, they make it possible to avoid lengthy and subjective setting of parameters by an expert engineer. Nevertheless, the use of an unbalanced dataset can lead to unexpected, although traceable errors. The solution is to collect a dataset large enough to enable both learning and verification of proper functionality

Abundance, Depth Distribution, and Composition of Aerobic Bacteriochlorophyll a-Producing Bacteria in Four Basins of the Central Baltic Sea▿ †

The abundance, vertical distribution, and diversity of aerobic anoxygenic phototrophic bacteria (AAP) were studied at four basins of the Baltic Sea. AAP were enumerated by infrared epifluorescence microscopy, and their diversity was analyzed by using pufM gene clone libraries. In addition, numbers of CFU containing the pufM gene were determined, and representative strains were isolated. Both approaches indicated that AAP reached maximal abundance in the euphotic zone. Maximal AAP abundance was 2.5 × 105 cells ml−1 (11% of total prokaryotes) or 1.0 × 103 CFU ml−1 (9 to 10% of total CFU). Environmental pufM clone sequences were grouped into 11 operational taxonomic units phylogenetically related to cultivated members of the Alpha-, Beta-, and Gammaproteobacteria. In spite of varying pufM compositions, five clones were present in all libraries. Of these, Jannaschia-related clones were always found in relative abundances representing 25 to 30% of the total AAP clones. The abundances of the other clones varied. Clones potentially affiliated with typical freshwater Betaproteobacteria sequences were present at three Baltic Sea stations, whereas clones grouping with Loktanella represented 40% of the total cell numbers in the Gotland Basin. For three alphaproteobacterial clones, probable pufM phylogenetic relationships were supported by 16S rRNA gene analyses of Baltic AAP isolates, which showed nearly identical pufM sequences. Our data indicate that the studied AAP assemblages represented a mixture of marine and freshwater taxa, thus characterizing the Baltic Sea as a “melting pot” of abundant, polyphyletic aerobic photoheterotrophic bacteria

Seasonal changes and diversity of aerobic anoxygenic phototrophs in the Baltic Sea

The community of aerobic anoxygenic phototrophs was investigated in the Baltic Sea using infrared epifluorescence microscopy from September 2004 to October 2005. The majority of these bacteriochlorophyll-containing organisms exhibited a specific sickle-shaped morphology, with rods or other morphotypes observed only occasionally. Aerobic anoxygenic phototrophs were observed mostly from April to September (1 to 12% of total prokaryotes), peaking in May 2005 at a concentration of up to 0.38 × 106 cells ml–1. This peak was associated with the later phase of the spring bloom. In the later months, the amount of phototrophic bacteria gradually declined until the beginning of the fall mixing, and remained low from November to March, contributing only 0 to 2% of total prokaryotes. A novel technique combining fluorescent in situ hybridization (FISH) and infrared epifluorescence microscopy indicated that the Baltic aerobic anoxygenic phototrophs were mostly Gammaproteobacteria, with a smaller fraction of Alphaproteobacteria

Decelularizace prasečí karotidy:Od cévy k vysoce kvalitnímu skafoldu za pět hodin

Využití biologicky odvozených cév jako cévních štěpů malého průměru u cévních onemocnění je v současnosti intenzivně studováno. Decelularizace cév poskytuje biokompatibilitu skeletu s velmi nízkou imunogenicitou, který zabraňuje imunosupresi po transplantaci. Dobré uchování skafoldu je důležité, protože usnadňuje úspěšnou buněčnou repopulaci. Kromě toho je třeba pečlivě vyhodnotit mechanické vlastnosti. Pokud je štěp zamýšlen pro použití jako tepna bude vystaven vysokým tlakům. Představujeme nový a rychlý decelularizační protokol pro prasečí karotidy, následovaný zkoumáním kvality získaných cévních skafoldů z hlediska zachování důležitých složek extracelulární matrice, mechanické odolnosti a kompatibility s lidskými endoteliálními buňkami. Naše výsledky dokazují, že náš protokol decelularizace minimálně mění řítomnost skafoldových proteinů i jejich mechanické chování a lidské endoteliální buňky by tak mohly adherovat na skafold in vitro. Dospěli jsme k závěru, že při použití vhodného protokolu lze rychle získat vysoce kvalitní decelularizované arteriální skafold jiného než lidského původu, který má velký potenciál k recelularizaci a použití jako arteriální štěp v transplantační medicíně.The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine

Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images

Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy

MicrAnt: Towards Regression Task Oriented Annotation Tool for Microscopic Image

Annotating a dataset for training a Supervised Machine Learning algorithm is time and annotator’s attention intensive. Our goal was to create a tool that would enable us to create annotations of the dataset with minimal demands on expert’s time. Inspired by applications such as Tinder, we have created an annotation tool for describing microscopic images. A graphical user interface is used to select from a couple of images the one with the higher value of the examined parameter. Two experiments were performed. The first compares the speed of annotation of our application with the commonly used tool for processing microscopic images. In the second experiment, the texture description was compared with the annotations from MicrAnt application and commonly used application. The results showed that the processing time using our application is 3 times lower and the Spearman coefficient increases by 0.05 than using a commonly used application. In an experiment, we have shown that the annotations processed using our application increase the correlation of the studied parameter and texture descriptors compared with manual annotations

Experimental fortification of intestinal anastomoses with nanofibrous materials in a large animal model

Anastomotic leakage is a severe complication in gastrointestinal surgery. It is often a reason for reoperation together with intestinal passage blockage due to formation of peritoneal adhesions. Different materials as local prevention of these complications have been studied, none of which are nowadays routinely used in clinical practice. Nanofabrics created proved to promote healing with their structure similar to extracellular matrix. We decided to study their impact on anastomotic healing and formation of peritoneal adhesions. We performed an experiment on 24 piglets. We constructed 3 hand sutured end-to-end anastomoses on the small intestine of each pig. We covered the anastomoses with a sheet of polycaprolactone nanomaterial in the first experimental group, with a sheet of copolymer of polylactic acid with polycaprolactone in the second one and no fortifying material was used in the Control group. The animals were sacrificed after 3 weeks of observation. Clinical, biochemical and macroscopic signs of anastomotic leakage or intestinal obstruction were monitored, the quality of the scar tissue was assessed histologically, and a newly developed scoring system was employed to evaluate the presence of adhesions. The material is easy to manipulate with. There was no mortality or major morbidity in our groups. No statistical difference was found inbetween the groups in the matter of level of peritoneal adhesions or the quality of the anastomoses. We created a new adhesion scoring system. The material appears to be safe however needs to be studied further to prove itsʹ positive effects