Selective cell response on natural polymer bio-interfaces textured by femtosecond laser

This study reports on the evaluation of laser processed natural polymer-chitosan, which is under consideration as a biointerface used for temporary applications as skin and cartilage substitutes. It is employed for tissue engineering purposes, since it possesses a significant degree of biocompatibility and biodegradability. Chitosan-based thin films were processed by femtosecond laser radiation to enhance the surface properties of the material. Various geometry patterns were produced on polymer surfaces and employed to examine cellular adhesion and orientation. The topography of the modified zones was observed using scanning electron microscopy and confocal microscopy. Test of the material cytotoxicity was performed by evaluating the life/dead cell correlation. The obtained results showed that texturing with femtosecond laser pulses is appropriate method to initiate a predefined cellular response. Formation of surface modifications in the form of foams with an expansion of the material was created under laser irradiation with a number of applied laser pulses from N = 1-5. It is shown that irradiation with N > 5 results in disturbance of microfoam. Material characterization reveals a decrease in water contact angle values after laser irradiation of chitosan films. Consequently, changes in surface roughness of chitosan thin-film surface result in its functionalization. Cultivation of MC3T3 and ATMSC cells show cell orientational migration concerning different surface patterning. The influence of various pulse durations (varying from tau = 30-500 fs) over biofilms surface was examined regarding the evolution of surface morphology. The goal of this study was to define the optimal laser conditions (laser energy, number of applied pulses, and pulse duration) to alter surface wettability properties and porosity to improve material performance. The acquired set of results indicate the way to tune the surface properties to optimize cell-interface interaction

ОПРЕДЕЛЯНЕ А ЕКСПРЕСИЯТА НА ФОСФАТИДИЛСЕРИН ПРИ СПЕРМАТОЗОИДИ В РАННА АПОПТОЗА ЧРЕЗ ФЛУОРЕСЦЕНТНО БИЛЯЗАН АНЕКСИН V

Double staining kit of Annexin V Cy3.18/6-CFDA was used to investigate the changes in phospholipide asymmetry after treating sperm cells with dexamethasone. The % of spermatozoa with registered translocation of PS in treated with dexamethazone groups at the 10-th min and in control no treated varied from 2.74%±0.65 to 2.30%±0.89. After the 5 hour of incubation these % increased to 39.83±3.33 for the treated group and 23.44±1.12 for the control. It was concluded that Annexin V binding assay is more sensitive in the detection of deterioration in membrane function than other conventional methods such as motility analysis and supravital techniques.Приложен е кит за двойно оцветяване с Анексин V Cy3.18 и 6-CFDA, даващ възможност за диференциране на апоптични от мъртви клетки. Процентът сперматозоиди с регистрирана експресия на ФС при свежи нетретирани сперматозоиди – контролна проба и експериментални, третирани с дексаметазон на 10-тата минута, варира от 2.30%±0.89 до 2.74%±0.65. След съхранение, на 5-я час този процент нараства до 23.44±1.12 за контролата и до 39.83±3.33 за опитната група. С настоящето изследване се доказва, че Анексин V теста е по-чувствителен за определяне промените в ПМ, в сравнение с конвенционалните методи на изследване за подвижност и преживяемост на сперматозоидите

CELL DEATH DIFFERENTIATION IN BLACK HEADED RAMS SPERMATOZOA, USING FLUORESCENT LABELED ANNEXIN V

Double staining kit of Annexin V Cy3.18/6-CFDA was used to investigate the changes in phospholipide asymmetry after treating sperm cells with dexamethasone. The % of spermatozoa with registered translocation of PS in treated with dexamethazone groups at the 10-th min and in control no treated varied from 2.74%±0.65 to 2.30%±0.89. After the 5 hour of incubation these % increased to 39.83±3.33 for the treated group and 23.44±1.12 for the control. It was concluded that Annexin V binding assay is more sensitive in the detection of deterioration in membrane function than other conventional methods such as motility analysis and supravital techniques

Improving osteoblasts cells proliferation via femtosecond laser surface modification of 3D-printed poly-ε-caprolactone scaffolds for bone tissue engineering applications

Synthetic polymer biomaterials incorporating cells are a promising technique for treatment of orthopedic injuries. To enhance the integration of biomaterials into the human body, additional functionalization of the scaffold surface should be carried out that would assist one in mimicking the natural cellular environment. In this study, we examined poly-epsilon-caprolactone (PCL) fiber matrices in view of optimizing the porous properties of the constructs. Altering the porosity of a PCL scaffold is expected to improve the material's biocompatibility, thus influencing its osteoconductivity and osteointegration. We produced 3D poly-epsilon-caprolactone (PCL) matrices by a fused deposition modeling method for bone and cartilage tissue engineering and performed femtosecond (fs) laser modification experiments to improve the surface properties of the PCL construct. Femtosecond laser processing is one of the useful tools for creating a vast diversity of surface patterns with reproducibility and precision. The processed surface of the PCL matrix was examined to follow the effect of the laser parameters, namely the laser pulse energy and repetition rate and the number (N) of applied pulses. The modified zones were characterized by scanning electron microscopy (SEM), confocal microscopy, X-ray computed tomography and contact angle measurements. The results obtained demonstrated changes in the morphology of the processed surface. A decrease in the water contact angle was also seen after fs laser processing of fiber meshes. Our work demonstrated that a precise control of material surface properties could be achieved by applying a different number of laser pulses at various laser fluence values. We concluded that the structural features of the matrix remain unaffected and can be successfully modified through laser postmodification. The cells tests indicated that the micro-modifications created induced MG63 and MC3T3 osteoblast cellular orientation. The analysis of the MG63 and MC3T3 osteoblast attachment suggested regulation of cells volume migration

Estimation of errors in text and data processing

The company Adiss Lab Lts. obtained 1 000 000 medical reports that are either in free form text, or in XML format. One of the main goals of their development is to integrate an algorithm for information extraction (IE) in their platform. The verification of the algorithm’s output for a report is done by a medical doctor (MD) for a certain fee. Validating the correctness of all data would be overwhelming and very expensive. Hence, the problem, as presented by the company, is to provide a method (algorithm) which determines the minimum amount of reports that will validate the correctness of the IE algorithm and a procedure for selecting these reports. In order to solve the problem we have considered an algorithm-centric approach uses active learning and semi-supervised learning

Landscape-scale forest loss as a catalyst of population and biodiversity change

The BioTIME database was supported by ERC AdG BioTIME 250189 and ERC PoC BioCHANGE 727440. We thank the ERC projects BioTIME and BioCHANGE for supporting the initial data synthesis work that led to this study, and the Leverhulme Centre for Anthropocene Biodiversity for continued funding of the database. Also supported by a Carnegie-Caledonian PhD Scholarship and NERC doctoral training partnership grant NE/L002558/1 (G.N.D.), a Leverhulme Fellowship and the Leverhulme Centre for Anthropocene Biodiversity (M.D.), Leverhulme Project Grant RPG-2019-402 (A.E.M. and M.D.), and the German Centre of Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig (funded by the German Research Foundation; FZT 118, S.A.B.).Global biodiversity assessments have highlighted land-use change as a key driver of biodiversity change. However, there is little empirical evidence of how habitat transformations such as forest loss and gain are reshaping biodiversity over time. We quantified how change in forest cover has influenced temporal shifts in populations and ecological assemblages from 6090 globally distributed time series across six taxonomic groups. We found that local-scale increases and decreases in abundance, species richness, and temporal species replacement (turnover) were intensified by as much as 48% after forest loss. Temporal lags in population- and assemblage-level shifts after forest loss extended up to 50 years and increased with species’ generation time. Our findings that forest loss catalyzes population and biodiversity change emphasize the complex biotic consequences of land-use change.PostprintPeer reviewe

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

<p>Abstract</p> <p>Background</p> <p>Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.</p> <p>Results</p> <p>The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in <it>N. benthamiana </it>L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated <it>in vitro </it>by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed <it>E. coli </it>enterotoxin B subunit:<it>H. sapiens </it>mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing <it>Y. enterocolitica </it>UDP-GlcNAc 4-epimerase gene and <it>C. elegans </it>UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.</p> <p>Conclusion</p> <p>Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.</p

ОПРЕДЕЛЯНЕ А ЕКСПРЕСИЯТА НА ФОСФАТИДИЛСЕРИН ПРИ СПЕРМАТОЗОИДИ В РАННА АПОПТОЗА ЧРЕЗ ФЛУОРЕСЦЕНТНО БИЛЯЗАН АНЕКСИН V

Double staining kit of Annexin V Cy3.18/6-CFDA was used to investigate the changes in phospholipide asymmetry after treating sperm cells with dexamethasone. The % of spermatozoa with registered translocation of PS in treated with dexamethazone groups at the 10-th min and in control no treated varied from 2.74%±0.65 to 2.30%±0.89. After the 5 hour of incubation these % increased to 39.83±3.33 for the treated group and 23.44±1.12 for the control. It was concluded that Annexin V binding assay is more sensitive in the detection of deterioration in membrane function than other conventional methods such as motility analysis and supravital techniques.Приложен е кит за двойно оцветяване с Анексин V Cy3.18 и 6-CFDA, даващ възможност за диференциране на апоптични от мъртви клетки. Процентът сперматозоиди с регистрирана експресия на ФС при свежи нетретирани сперматозоиди – контролна проба и експериментални, третирани с дексаметазон на 10-тата минута, варира от 2.30%±0.89 до 2.74%±0.65. След съхранение, на 5-я час този процент нараства до 23.44±1.12 за контролата и до 39.83±3.33 за опитната група. С настоящето изследване се доказва, че Анексин V теста е по-чувствителен за определяне промените в ПМ, в сравнение с конвенционалните методи на изследване за подвижност и преживяемост на сперматозоидите