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Background Graves’ disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves’ orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (βgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). Results In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-βgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors’ microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25⁺ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. Conclusions These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments.

On-farm culture (OFC) systems facilitate pathogen-based mastitis management and can facilitate antimicrobial stewardship on dairy farms. Interpretation of the results, however, may present a challenge for those with limited microbiology experience. Here, we compared results of 3 OFC systems interpreted by trained and untrained observers against results of a standard laboratory reference method (aerobic culture and mass spectrometry). Milk samples (280 quarter and 60 composite) were selected from submissions for routine diagnostic testing to Quality Milk Production Services (Cornell University, Ithaca, NY) between August 2017 and January 2018. Samples were cultured simultaneously using the standard laboratory reference method and 3 commercially available OFC systems that varied in detail of pathogen identification (provided in parentheses) as follows: (1) Minnesota Easy Culture System II Bi-plate (University of Minnesota Laboratory for Udder Health, St. Paul; gram-positive, gram-negative), (2) Minnesota Easy Culture System II Tri-plate (gram-positive, gram-negative, some genus level), and (3) FERA Diagnostics and Biologicals AccuMast plate (Ithaca, NY; genus level, some species level). After 18 to 24 h of incubation, OFC plates were interpreted by 1 trained observer (>10 yr of experience in milk microbiology) and 6 untrained observers with no previous milk microbiology training, using only the manufacturers' instructions for guidance. Strength of agreement (?) between observer groups and the reference method was determined for the available outcomes of each system. Interpreted by the trained observer, agreement was moderate for identifying gram-positive organisms (Bi-plate, ? = 0.56) and substantial for Streptococcus spp. (Tri-plate, ? = 0.64, AccuMast ? = 0.61). Interpretation by untrained observers resulted in fair agreement (? = 0.29-0.37) for these organisms. Moderate agreement (? = 0.43-0.59) was found across all 3 OFC for the identification of gram-negative organisms (Bi-plate), non-aureus staphylococci (Tri-plate and AccuMast), Lactococcus spp., and Enterococcus spp. (AccuMast) when interpreted by the trained observer, and fair to moderate agreement was found (? = 0.31-0.53) among untrained observers. Across all 3 OFC, agreement was almost perfect (? = 0.80-0.89) for Staphylococcus aureus for the trained observer, and moderate to substantial (? = 0.56-0.61) for untrained observers. We concluded that all 3 OFC appeared suitable to support pathogen-based mastitis management when operated by trained observers. Training beyond the instruction manual is a prerequisite to make OFC systems useful for pathogen-based mastitis management.

Il sequenziamento del genoma canino ha contribuito a migliorare l’accuratezza delle stime di diversità genetica ed ha anche generato diversi studi sulle relazioni tra le diverse razze allevate (Vaysse et al., 2011; Parker et al., 2017). Tuttavia, alcune razze rimangono ancora poco caratterizzate. È il caso del Bracco Francese tipo Pyrénées. Lo scopo di questo lavoro è stato quello di caratterizzare a livello genomico la razza e studiarne le relazioni con altre razze allevate nel mondo. Sono stati raccolti i campioni di sangue da 48 individui. La genotipizzazione è stata condotta mediante l’Illumina CanineHD BeadChip (173.662 marcatori). Diversi parametri di diversità genetica sono stati calcolati, come l’ eterozigosià osservata (Ho) ed attesa (He). La consanguineità (F) è stata stimata con il metodo delle Runs of homozygosity (ROH). La relazione genetica tra il Bracco Francese e altre razze canine è stata studiata utilizzando i dati genotipici di 532 individui appartenenti a 48 razze canine e 15 lupi, ottenuti dal progetto LUPA (Vaysse et al., 2011). È stata costruita una matrice di distanze genetiche per generare un albero filogenetico (neighbor joining). Il controllo di qualità dei dati genotipici, la stima degli indici di diversità genetica, il rilevamento dei segmenti ROH e la stima delle distanze genetiche sono state condotte utilizzando il software PLINK (Purcell et al., 2007). I valori medi di Ho e He erano di 0,371±0,142 e 0,359±0,124. La consanguineità media stimata su base ROH (FROH) era di 0,112±0,023. Comparando questi valori con quelli riportati in letteratura, è emerso che la razza conserva ancora sufficienti livelli di variabilità genetica, utili per predisporre piani di conservazione e di miglioramento genetico. Il multidimensional scaling (MDS) della matrice di distanze genetiche (C1) (Figura 1) ha rivelato tre cluster principali, con il Bracco Francese (BRA) altamente differenziato dalle altre razze. In particolare, la maggior parte delle moderne razze europee erano raggruppate insieme e parzialmente sovrapposte nel centro del grafico, suggerendo un flusso genico relativamente alto. Si ritiene che il BRA sia imparentato con altre razze da ferma europee, ma la relazione esatta tra queste razze rimane poco chiara Sulla base dei risultati qui riportati, alcune razze come Weimaraner (WEI), Setter Inglese (EST) e Gordon Setter e (GOS) sembrano essere geneticamente più vicine al BRA. Simili risultati sono evidenziati in Figura 2, dove l'albero filogenetico ha confermato la stretta relazione tra BRA, EST e GOS. L'albero ha anche mostrato che le razze con caratteristiche fenotipiche simili tendono a raggrupparsi insieme, come i retriever, setter, spaniel e terrier.

The gut microbiota-brain axis is a bidirectional pathway through which the brain regulates the activity of the gutand vice versa. The intestinal microbiota was proven to influence and be influenced by stress-related responsescaused by management conditions. The aim of this work was to evaluate whether different husbandry conditionsin lambs influenced gut microbiome (composition and diversity), cortisol level and behaviour. Fifteen Sarda breedlambs, aged 6 months, were randomly assigned to three groups of 5 animals each: Isolation (tactile and visualisolation; 3 h/day for 8 weeks); Enriched (in group in a outdoor grassy pen with a new enrichment each week; 3h/day for 8 weeks); Control (habitual farm environment). Every week, 2 video-recordings of 1-hour each werecollected respectively during and after each treatment, and analysed using a scan sampling technique (60 scansper video). Every two weeks hair was collected from the right shoulder using a shave and re-shave method.Cortisol concentration was measured by means of an ELISA test conducted on hair samples and expressed aspg/ml. At week 8 faecal material was collected directly from the rectal ampulla and immediately frozen at -20°Cuntil DNA extraction was performed using a QIAmp DNA Stool kit (Qiagen, Hilden, Germany). DNA quality andquantity were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE,USA). The microbiota of enriched animalswas clearly different from the other two groups and showed lowerwithin group variability. During treatment, only isolated sheep showed escape attempts (23% of scans during thefirst week) and freezing (58% of scans during the first week). In the home pen, after treatment, lambs did not showany stress-related behaviour. After one month of treatment, hair cortisol increased only in the isolated group. Inconclusion, differences in microbiome could be related to the different stimulating enrichment and addition ofgrass on the diet. Although, from a behavioural point of view, sheep apparently habituated to the isolation,increase in cortisol levels could be related to experiencing a stressful situation. This study was approved by theItalian National Ethical Commission (authorization n°457/2016-PR) and supported by MIUR-PRIN2015 (Grant2015Y5W9YP).

A series of hydrophilic alkanethiols, HS(CH2)(n)OH with n = 3, 4, 6, 8, 9 and 11, have been self-assembled on gold electrodes of pentacene-based thin-film transistors. The multiparametric and ultra-sensitive response of these devices allows us to characterize both charge-injection and electrical stability moving from vacuum to air. The decay exponent beta of charge injection is found to be 0.7-1.1 angstrom(-1) in agreement with earlier measurements of charge tunneling done by electrochemistry and large-area molecular junctions. We find that the intermediate chain lengths (n = 4 and 6) yield an optimum response as they represent the best compromise in terms of decrease of the contact-resistance and bias stress.

The increasing availability of SNP (single nucleotide polymor- phisms) genotype data in livestock is stimulating the develop- ment of new data analysis strategies, which can be applied in animal breeding. One possible application is the prediction of carriers of specific haplotypes, especially if they impact animal health. It is therefore convenient to have a practical and easy-to- implement statistical method for the accurate classification of individuals into carriers and non-carriers. In this paper, we pres- ent a procedure for the identification of carriers of the haplotype HH1 on BTA5 (Bos Taurus autosome 5), which is known to be associated with reduced cow fertility in Holstein-Friesian cattle. A population of 1104 Holstein bulls genotyped with the 54K SNP- chip was available for the analysis. There were 45 carriers (5.3%) and 1045 non-carriers (94.7%). Two complementary mul- tivariate statistical techniques were used for the identification of haplotype carriers: Backward Stepwise Selection (BSS) to select the SNP that best fit the model, and Linear Discriminant Analysis (LDA) to classify observations, based on the selected SNP, into carriers and non-carriers. In order to explore the min- imum-sized set of SNP that correctly identifies haplotype carri- ers, different proportions of SNP were tested: 2.5; 10; 15; 30; 50 and 100%. For each proportion of SNP, BSS and LDA were applied, and the classification error rate was estimated in a 10- fold cross-validation scheme. Data were split in 10 subsets. The first subset was treated as validation set, while the model was fit on the remaining nine subsets (the training set). The overall error rate for the prediction of haplotype carriers was on average very low (~1%) both in the training and in the validation datasets. The error rate was found to depend on the number of SNPs in the model and their density around the region of the haplotype on BTA5. The minimum set of SNPs to achieve accu- rate predictions was 8, with a total test error rate of 1.27. This work describes a procedure to accurately identify haplotype car- riers from SNP genotypes in cattle populations. Very few misclas- sifications were observed, which indicates that this is a very reli- able approach for potential applications in cattle breeding.

In the last decade nanotechnology has allowed to achieve important improvements in designing novel materials, by controlling their properties over the nanometer scale, and then in integrating them into devices. In the life science domain, nanotechnology are expected to play a key role in developing new diagnostic methods, loco-regional therapies and, in regenerative medicine, scaffold for artificial tissues and organs. Accurate control of cell adhesion is becoming a key issue in domain. Soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [1] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. The combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art substrates with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. An application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues will be presented. Patterns of biomolecules and proteins have been fabricated, by a modified Lithographically Controlled Wetting (LCW), on the highly antifouling surface of Teflon-AF to guide the adhesion, growth and differentiation of neural cells (SHSY5Y, 1321N1, NE-4C) achieving an extremely accurate guidance [2]. A straightforward approach for the control of cell fate is based on the realization of neighboring surface features displaying opposite cell anchoring properties, the coexistence of surface regions favoring the cell adhesion with antifouling ones will provide a strong guidance for cell growth and migration. In this frame, Teflon is an interesting material, already employed in medical tools and devices (catheters, stents), is biocompatible, inert but at the same time strongly unprone to cell adhesion. Local surface topography is also known to influence the cell fate [3], thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. In this perspective we have developed a novel fabrication technique, named Lithographically controlled Etching (LCE), allowing, in one step, to engrave and to functionalize the substrate surface over different length scales and with different functionalities. Our technological approach has the advantages to be an one-step procedure, without background passivation steps and chemical modifications of the substrates, suitable for controlling cell guidance. The results show a viable route for creating cell arrays or multi-layers cell architectures to be used in cell biology, pharmacological and toxicological studies and tissue engineering.

This presentation deals with a modellization developed for describing OTFTs' instability upon bias stress effect through the analysis of hysteretic transfer characteristics

Data on performance of animals are, in several situations, collected at the group rather than individual level. Genetic evaluations in farm animals, however, are based on phenotypic information collected at the individual level. Therefore, it would be very attractive to extend genetic evaluations by incorporating information collected at the group level. In this paper we show the use of data collected at the group level for the estimation of variance components and the prediction of breeding values. We outline a general procedure that can be applied in different farm animal species. In the present work this procedure was applied to BW, for which pooled, as well as individual, observations were available, thus allowing for a comparison of the estimates, and to egg production, for which only pooled data were available. For BW at 19 and 27 wk the estimated heritabilities based on individual observations were very similar to those based on pooled observations. For BW at 43 and 51 wk, heritability estimates based on individual and pooled data were different, which can be caused by the emergence of competition effects. The accuracy of EBV predicted from pooled observations was about 70 to 80% of the accuracy of EBV predicted from individual observations. This result quantifies the loss deriving from the use of pooled instead of individual observations. Results show that estimation of variance components and breeding values from pooled data instead of individual observations is theoretically and practically feasible.

We report adhesion, growth, and differentiation of mouse neural cells on ultra-thin films of an organic semiconductor, pentacene. We demonstrate that i) pentacene is structurally and morphologically stable upon prolonged contact with water, physiological buffer, and cell culture medium; ii) neural stem cells adhere to pentacene and remain viable on it for at least 15 days; iii) densely interconnected neural networks and glial cells develop on the pentacene surface after several days. This implies that adhesion proteins secreted by the cells find suitable adsorption loci to anchor the cells. Pentacene is also a suitable substrate for casting thin layers of cell adhesion molecules, such as laminin and poly-l-lysine. Our results show that pentacene, albeit being an aromatic molecule, allows neurons to adhere to and grow on it, which is possibly due to its tightly packed solid state structure. This structure remains unaltered upon exposure to water and interfacial force exerted by the cells. The integration of living cells into organic semiconductors is an important step towards the development of bio-organic electronic transducers of cellular signals from neural networks.

Many technological advances are currently being developed for nano- and micro- fabrication, offering the ability to create and control patterns of soft materials with sub micrometric precision. In this work we report the deposition of cells on organic semiconductor ultra-thin films. This is a first step towards the development of active bio/non bio systems for electrical transduction that could be integrated directly in vitro. Thin films of pentacene molecules, whose thickness was systematically varied between 1 and 50 nm, were grown by high vacuum sublimation. Human astrocytoma cells and NE-4C neural stem cells were grown and maintained in culture in standard condition (37oC, 95% humidity, 5%CO2) in medium supplemented. Small molecules (PL, laminine, APTES, dodecylamine, PEG etc.) were patterned onto the surface by template-assisted deposition in order to spatially modulate the cell adhesion on the surface. Released pre-patterned multilayers were exposed to cell culture medium incubated at 37oC for 70h. Viability of cells in time was measured as a function of roughness and the characteristic morphology of ultra thin organic film, as well as the features of the patterned molecules. Optical fluorescence microscope coupled to atomic force microscope was used to monitor the presence, density and shape of deposited cells. Acknowledgements This work is supported by Project EU-NMP-STRP 032652 BIODOT.

The growth of vacuum-sublimed tetracene thin films on silicon dioxide has been investigated from the early stages of the process. The effects of deposition flux and substrate silanization on film morphology and electrical properties have been explored. Tetracene shows an island growth, resulting in films with a granular structure. Both an increase in the deposition flux and the substrate silanization determine a decrease of the grain size and an improvement of the connectivity of the film in direct contact with the substrate. The hole mobility in field-effect transistors based on tetracene thin films, which also generate electroluminescence, increases with the deposition flux and values as high as 0.15 cm(2) V(-1) s(-1) are obtained.