Carcass and meat quality in growing-finishing pigs fed diets with plant-based protein sources alternative to genetically modified soybean meal

The objective of this study was to evaluate carcass and meat quality in growing-finishing pigs fed diets with different vegetable protein sources. It was found that partial (50% in grower diets) and complete (100% in finisher diets) replacement of protein from genetically modified soybean meal (GM-SBM) with protein from 00-rapeseed meal (00-RSM), alone or in combination with protein from faba bean seeds (FB) cv. ‘Albus’, yellow lupine seeds (YL) cv. ‘Taper’ or corn DDGS, had no significant effect on carcass quality characteristics or the chemical composition of meat. In all groups, meat samples were characterized by color typical of pork, high water-holding capacity and low pH values. A sensory analysis of the eating quality attributes of meat revealed that they were highly satisfactory; only aroma intensity was affected by the experimental factor. The study demonstrated that growing-finishing pigs can be fed complete diets containing the analyzed vegetable protein sources alternative to GM-SBM without compromising carcass or meat quality

In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3®

Introduction. Primary cells in vitro culture scale-up is a crucial issue in cell-based tissue and organ regeneration therapy. Reducing costs and space occupied by the cells cultured in vitro has been an important target. Cells cultured in vitro with the use of bioreactor with dextran microcarriers (Cytodex®) have potentially a chance to meet many of the cell therapy requirements. Material and methods. We used collagen-coated carriers (Cytodex3®) and a spinner flask bioreactor to develop environment suitable for human myoblast proliferation. In parallel, standard adherent in vitro culture conditions for myoblasts propagation (T-flask) were conducted. Cell cycle characterization, senescence, myogenic gene ex­pression and cell apoptosis were evaluated in order to find differences between two culture systems under study. Results. The number of cells obtained in bioreactor per 106 of starting cells population was approximately ten times lower in comparison with T-flask culture system. The microcarriers cultured adult myoblasts in compari­son with the regular T-flask culture showed faster and more advanced replicative aging and lower proliferative potential. Moreover, the percentage of the cells that entailed an irreversible cell arrest (G0 phase) was also significantly (p < 0.0001) increased. Conclusions. Our results suggest that population of primary human myoblasts obtained from adult individuals and propagated on dextran microcarriers did not meet the requirements of the regenerative medicine regarding quantity and quality of the cells obtained. Nonetheless, further optimization of the cell scaling up process including both microcarriers and/or bioreactor program is still an important option

Mn-based methacrylated gellan gum hydrogels for MRI-guided cell delivery and imaging

This work aims to engineer a new stable injectable Mn-based methacrylated gellan gum (Mn/GG-MA) hydrogel for real-time monitored cell delivery into the central nervous system. To enable the hydrogel visualization under Magnetic Resonance Imaging (MRI), GG-MA solutions were supplemented with paramagnetic Mn2+ ions before its ionic crosslink with artificial cerebrospinal fluid (aCSF). The resulting formulations were stable, detectable by T1-weighted MRI scans and also injectable. Cell-laden hydrogels were prepared using the Mn/GG-MA formulations, extruded into aCSF for crosslink, and after 7 days of culture, the encapsulated human adipose-derived stem cells remained viable, as assessed by Live/Dead assay. In vivo tests, using double mutant MBPshi/shi/rag2 immunocompromised mice, showed that the injection of Mn/GG-MA solutions resulted in a continuous and traceable hydrogel, visible on MRI scans. Summing up, the developed formulations are suitable for both non-invasive cell delivery techniques and image-guided neurointerventions, paving the way for new therapeutic procedures.Sílvia Vieira acknowledges the FCT Ph.D. scholarship (SFRH/BD/102710/2014). J. Miguel Oliveira and J. Silva-Correia acknowledge the FCT grants under the Investigator FCT program (IF/01285/2015 and IF/00115/2015, respectively). The authors also acknowledge the funds provided under the project NanoTech4ALS, funded under the EU FP7 M-ERA.NET program, and ESF (POWR.03.02.00-00-I028/17-00)

Der Einfluss der Mikrogravitation und der Strömung auf die Regulation der E3 Ligase NEDD4 und ihr Substrat Cx43 in Endothelzellen

The reduction of capillary density in microgravity-induced muscle atrophy has been observed by several studies. The cellular and molecular mechanisms leading to these changes remain to be recognized. Therefore, this study was designed at testing endothelial reactions on microgravity, focussing on those which are probably connected to the regulation of blood vessel growth. Since, according to published results from a gene array analysis, one of the most strongly regulated genes by microgravity is E3-ligase NEDD4, the aim of this work was to validate the impact of microgravity and shear stress (as a side effect when producing microgravity) on it, and search for NEDD4-regulated, angiogenesis-related proteins. In addition, previous studies indicated that ADAMTS1, which plays a role in the regulation of angiogenesis, is strongly induced by mechanical forces acting on endothelial cells, namely by shear stress. Here, it was tested if there is any influence of microgravity on ADAMTS1 expression. HUVEC and EAhy926 cells were exposed to constant laminar or pulsatile laminar flow in a cone and plate device developed in the institute´s workshop. Microgravity experiments were performed in a Rotating Wall Vessel bioreactor. Both, NEDD4 and ADAMTS1, were tested under increased shear stress and microgravity. The regulation of genes and proteins was analysed by real time RT-PCR, immunoblotting, and immunoprecepitation, respectively. Additionally, cells were treated with different inhibitors either to block protein degradation or to investigate the role of mechanosensitive signalling pathways in their regulation. NEDD4 is expressed in endothelial cells, co-precipitates with Cx43, and the increase of NEDD4 induced by microgravity correlates with a decrease in Cx43. This interaction between NEDD4 and Cx43 in human endothelial cells was confirmed by immunostaining showing co-localization and by siRNA mediated knockdown of NEDD4 which caused a decrease in Cx43. Accordingly, an increase of Cx43 was shown with inhibitors of the proteasome. However, there was no effect of constant laminar flow on NEDD4 expression. On contrary, an atheroprotective flow profile upregulated NEDD4 while an atheroprone flow profile caused the opposite effect. PI3K and NO˙ production did not play a role in the regulation of NEDD4 by shear stress, so other signalling pathways will be involved. ADAMTS1 was transiently induced by microgravity. Shear stress from 2 dyn/cm2 onwards induced ADAMTS1, whereas atheroprone flow reduced it. Secreted to the cell culture supernatant, ADAMTS1 cleaves TSP1 to its smaller anti-angiogenic fragments. PI3K and NO˙ production are clearly involved in the regulation of ADAMTS1 by shear stress. Likewise, TSP1 was somewhat later but as well transiently induced by microgravity. Taken together, this work supports earlier observations of reduced angiogenesis under microgravity and extends them towards additional cellular mechanisms which contribute to the anti- angiogenic phenotype of endothelial cells under these conditions. Thus, the results of this study are expected to provide better understanding of mechanisms which can contribute not only to the development of different pathological situation observed in astronauts, but also for patients during long bed rest and for those who suffer from atherosclerosis.Eine reduzierte Kapillardichte bei durch Mikrogravitation ausgelöster Muskelatrophie ist in mehreren Studien beschrieben worden. Dennoch sind die zellulären und molekularen Mechanismen, die zu diesem Verlusst an Kapillaren führen, bisher unbekannt. Diese Studie hatte daher zum Ziel, Reaktionen von Endothelzellen auf Mikrogravitation zu untersuchen, insbesondere solche, für die ein Zusammenhang mit der Regulation des Blutgefäßwachstums besteht. Eine Gene-array Analyse einer anderen Gruppe hatte zuvor gezeigt, dass in Endothelzellen die E3-Ligase NEDD4 besonders deutlich durch Mikrogravitation induziert wird. Daher sollten nun diese Ergebnisse mit anderen Methoden bestätigt und auf Cx43 als Angiogenese-assoziiertes Substrat von NEDD4 ausgedehnt werden. Darüber hinaus sollte untersucht werden, ob das mechanosensitiv induzierte ADAMTS1, ein Angiogeneseinhibitor, auch eine Expressionsregulation durch Mikrogravitation erfährt. Humane umbilikale venöse Endothelzellen und EAhy926 Zellen wurden in einem Konus-Platte-System konstanter laminarer oder pulsatil laminarer Strömung ausgesetzt. Mikrogravitation wurde mithilfe des von der NASA entwickelten „Rotating Wall Vessel“ simuliert. Anschließend wurden die Zellen mittels quantitativer real time RT-PCR, Immunoblotting und Immunpräzipitation untersucht. In weiteren Experimenten wurden die Zellen mit Inhibitoren des Proteasoms oder des Lysosoms, mit einem PI3K-Inhibitor oder L-NAME zur Hemmung der NO˙-Synthese behandelt, um einen etwaigen Beitrag dieser Signalwege zur beobachteten Regulationscharakteristik zu ermitteln. NEDD4 wird in Endothelzellen exprimiert, lässt sich mit Cx43 kopräzipitieren, und sein durch Mikrogravitation erzeugter Konzentrationsanstieg korrelliert mit dem Konzentrationsabfall von Cx43. Die Interaktion von NEDD4 und Cx43 wurde durch Kolokalisation in der konfokalen Lasermikroskopie sowie nach siRNA vermitteltem knock down von NEDD4 bestätigt. Entsprechend führte die Hemmung des Proteasoms zum Anstieg von Cx43. Konstante, laminare Strömung hatte dagegen keinen Einfluss auf die Expression von NEDD4. Wohl aber wurde NEDD4 durch ein atheroprotektives Strömungsprofil induziert, durch ein atherogenens supprimiert. PI3K und NO˙ spielten dabei keine Rolle. ADAMTS1 wurde durch Mikrogravitation transient induziert. Schubspannungen über 2 dyn/cm2 führten zu einem starken Anstieg von ADAMTS1, während es durch ein atherogenes Strömungsprofil supprimiert wurde. In den Kulturüberstand sezerniert degradiert ADAMTS1 TSP1 zu einem antiangiogenen, 70 kDa großen Fragment. PI3K und NO˙ Produktion sind gleichermaßen notwendig für die strömungsabhängige Expressionsregulation von ADAMTS1. Auch TSP1 wurde durch Mikrogravitation transient induziert. Diese Arbeit unterstützt die bereits bestehende Auffassung von einer reduzierten Angiogenese unter den Bedingungen der Schwerelosigkeit und erweitert sie um zelluläre/molekulare Mechanismen, die zur Erklärung des antiangiogenen Phänotyps der Endothelzellen bei Schwerelosigkeit beitragen können. Diese Ergebnisse tragen daher bei zu einem besseren Verständnis pathophysiologischer Vorgänge bei Astronauten (bzw. Kosmonauten u.ä.) und – klinisch relevanter – bei Patienten mit langer Immobilisation. Wegen der Rolle von Cx43 bei der Entwicklung von Arteriosklerose könnten die hier vorgelegten Ergebnisse über NEDD4 auch in diesem Zusammenhang von Interesse sein

Acyclic colourings of graphs with bounded degree

Graphs and AlgorithmsA k-colouring of a graph G is called acyclic if for every two distinct colours i and j, the subgraph induced in G by all the edges linking a vertex coloured with i and a vertex coloured with j is acyclic. In other words, there are no bichromatic alternating cycles. In 1999 Boiron et al. conjectured that a graph G with maximum degree at most 3 has an acyclic 2-colouring such that the set of vertices in each colour induces a subgraph with maximum degree at most 2. In this paper we prove this conjecture and show that such a colouring of a cubic graph can be determined in polynomial time. We also prove that it is an NP-complete problem to decide if a graph with maximum degree 4 has the above mentioned colouring

Cutaneous and Subcutaneous Tumours of Small Pet Mammals—Retrospective Study of 256 Cases (2014–2021)

Since small mammals are gaining popularity as pets in Poland, the number of tumour samples submitted for histopathological examination is quite high. This study was a retrospective analysis of cutaneous and subcutaneous tumours in small pet mammals submitted for histopathology in 2014–2021. The analysis included 256 tumours sampled from 103 guinea pigs, 53 rats, 43 pet rabbits, 21 ferrets, 17 hamsters, 8 degus, 5 African pygmy hedgehogs, 3 Mongolian gerbils and 3 chinchillas. Tumours were diagnosed based on routine histopathology, with additional immunohistochemistry when necessary. The results of this study revealed that the vast majority of cutaneous tumours in guinea pigs were benign, with a predominance of lipoma. Adnexal tumours constituted a significant percentage of cutaneous tumours in guinea pigs (24.3%, with the most common being trichofolliculoma), pet rabbits (46.5%, with the most common being trichoblastoma), ferrets (33.3%, mostly derived from sebaceous glands), hamsters (52.9%, with the most common being trichoepithelioma) and gerbils (66.7%, scent gland epithelioma). Soft tissue sarcomas were a predominant group of tumours in rats (52.8%, with the most common being fibrosarcoma), African pygmy hedgehogs (100%), degus (87.5%) and chinchillas (66.7%). Melanocytic tumours were only sporadically seen in small mammal pets. Mast cell tumours were diagnosed only in ferrets, while epitheliotropic T-cell lymphoma was diagnosed only in a hamster and a degu. In summary, malignant tumours constitute a significant percentage of cutaneous tumours in many species of small mammal pets. Therefore, each cutaneous tumour should be sampled for further cytologic or histopathologic diagnosis

Cutaneous and Subcutaneous Tumours of Small Pet Mammals—Retrospective Study of 256 Cases (2014–2021)

Since small mammals are gaining popularity as pets in Poland, the number of tumour samples submitted for histopathological examination is quite high. This study was a retrospective analysis of cutaneous and subcutaneous tumours in small pet mammals submitted for histopathology in 2014–2021. The analysis included 256 tumours sampled from 103 guinea pigs, 53 rats, 43 pet rabbits, 21 ferrets, 17 hamsters, 8 degus, 5 African pygmy hedgehogs, 3 Mongolian gerbils and 3 chinchillas. Tumours were diagnosed based on routine histopathology, with additional immunohistochemistry when necessary. The results of this study revealed that the vast majority of cutaneous tumours in guinea pigs were benign, with a predominance of lipoma. Adnexal tumours constituted a significant percentage of cutaneous tumours in guinea pigs (24.3%, with the most common being trichofolliculoma), pet rabbits (46.5%, with the most common being trichoblastoma), ferrets (33.3%, mostly derived from sebaceous glands), hamsters (52.9%, with the most common being trichoepithelioma) and gerbils (66.7%, scent gland epithelioma). Soft tissue sarcomas were a predominant group of tumours in rats (52.8%, with the most common being fibrosarcoma), African pygmy hedgehogs (100%), degus (87.5%) and chinchillas (66.7%). Melanocytic tumours were only sporadically seen in small mammal pets. Mast cell tumours were diagnosed only in ferrets, while epitheliotropic T-cell lymphoma was diagnosed only in a hamster and a degu. In summary, malignant tumours constitute a significant percentage of cutaneous tumours in many species of small mammal pets. Therefore, each cutaneous tumour should be sampled for further cytologic or histopathologic diagnosis

pNiPAM-Nanoparticle-Based Antiapoptotic Approach for Pro-Regenerative Capacity of Skeletal Myogenic Cells

The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells

Transplantation of Human Glial Progenitors to Immunodeficient Neonatal Mice with Amyotrophic Lateral Sclerosis (SOD1/rag2)

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease with no effective therapy. The neurodegenerative character of ALS was an appealing target for stem cell-based regenerative approaches. Different types of stem cells have been transplanted in both preclinical and clinical settings, but no convincing outcomes have been noted. Human glial restricted precursors (hGRPs) transplanted intraventricularly to neonatal, immunodeficient mice rescued lifespan of dysmyelinated mice. Intraspinal injection of hGRPs also provided benefits in the mouse model of ALS. Therefore, we have recently developed an immunodeficient model of ALS (double mutant SOD1/rag2), and, in this study, we tested the strategy previously used in dysmyelinated mice of intraventricular transplantation of hGRPs to immunodeficient mice. To maximize potential therapeutic benefits, the cells were implanted into neonates. We used magnetic resonance imaging to investigate the progression of neurodegeneration and therapeutic responses. A cohort of animals was devoted to survival assessment. Postmortem analysis included immunohistochemistry, Nissl staining, and Western blots. Cell transplantation was not associated with improved animal survival, slowing neurodegeneration, or accumulation of misfolded superoxide dismutase 1. Postmortem analysis did not reveal any surviving hGRPs. Grafting into neonatal immunodeficient recipients did not prevent ALS-induced cell loss, which might explain the lack of positive therapeutic effects. The results of this study are in line with the modest effects of clinical neurotransplantations. Therefore, we urge stem cell and ALS communities to develop and implement cell tracking methods to better understand cell fates in the clinic

Deconvolution of axisymmetric flame properties using Tikhonov regularization

Peer reviewed: YesNRC publication: Ye