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It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158–167, 2018.

Tomosynthesis (DBT) has proven to be more sensitive than digital mammography, but it requires longer reading time. We retrospectively compared accuracy and reading times of a simplified protocol with 1-cm-thick slabs versus a standard protocol of slabs + 1-mm-spaced planes, both integrated with synthetic 2D. We randomly selected 894 DBTs (including 12 cancers) from the experimental arm of the RETomo trial. DBTs were read by two radiologists to estimate specificity. A second set of 24 cancers (8 also present in the first set) mixed within 276 negative DBTs was read by two radiologists. In total, 28 cancers with 64 readings were used to estimate sensitivity. Radiologists read with both protocols separated by a 3-month washout. Only women that were positive at the screening reading were assessed. Variance was estimated taking into account repeated measures. Sensitivity was 82.8% (53/64, 95% confidence interval (95% CI) 67.2–92.2) and 90.6% (95% CI 80.2–95.8) with simplified and standard protocols, respectively. In the random screening setting, specificity was 97.9% (1727/1764, 95% CI 97.1–98.5) and 96.3% (95% CI 95.3–97.1), respectively. Inter-reader agreement was 0.68 and 0.54 with simplified and standard protocols, respectively. Median reading times with simplified protocol were 20% to 30% shorter than with standard protocol. A simplified protocol reduced reading time and false positives but may have a negative impact on sensitivity. • The adoption of digital breast tomosynthesis (DBT) in screening, more sensitive than mammography, could be limited by its potential effect on the radiologists’ workload, i.e., increased reading time and fatigue. • A DBT simplified protocol with slab only, compared to a standard protocol (slab plus planes) both integrated with synthetic 2D, reduced time and false positives but had a negative impact on sensitivity.

La période du gouvernement de Pascal Paoli, notamment dans les années 1764-1769, est cruciale dans l’histoire de la Corse. L’île est alors, en effet, un enjeu politique de portée européenne, car la domination de la république de Gênes est en déclin, et Choiseul convoite ce nouveau territoire, de crainte que cette place stratégique ne tombe entre les mains du roi de Grande-Bretagne. James Boswell joua un rôle fondamental dans la représentation littéraire de la Corse et la consolidation de la notoriété de Pascal Paoli en tant que champion du patriotisme et de la liberté. Influencé par l’admiration qu’il éprouvait alors envers Jean-Jacques Rousseau, Boswell alla rencontrer le chef des rebelles insulaires. Son récit de voyage, intitulé An Account of Corsica (1768), fut un véritable ‘bestseller’. Avant ce récit, la définition de la Corse tenait en deux lignes dans l’Encyclopédie de Paris. Après 1768, l’auteur de l’entrée ‘Corse’ dans l’Encyclopédie d’Yverdon puise abondamment dans l’ouvrage de Boswell. Comme le dit Jean Viviès, ‘An Account of Corsica s’est inséré dans le savoir des Lumières tel que l’exposent les diverses Encyclopédies’ (SVEC 245, p.468). Le présent ouvrage étudie l’évolution de la représentation littéraire de la Corse et de Paoli dans la première moitié du dix-huitième siècle, en replaçant dans son contexte le voyage de Boswell, et les diverses impressions des visiteurs britanniques du bref épisode ‘anglo-corse’ des années 1794-1796; le lien qui relie les deux périodes étant Pascal Paoli. Remerciments Introduction Chronologie 1725-1769 1. La naissance et l’épanouissement du mythe de Pascal Paoli (1730-1775) i. L’image de la Corse avant 1730 ii. Premiers signes de sympathie iii. La découverte de la Corse par les Français iv. Le voyage de Christopher Hervey en 1760 v. Les ‘Pèlerins de la Liberté’ et autres curieux vi. Le paysage et les hommes vus par les ‘Pèlerins de la Liberté’ vii. Pascal Paoli: un héros de Plutarque viii. Pascal Paoli inspire ‘les Muses de la Liberté’ (1769-1775) Chronologie 1789-1807 2. L’étoile pâlissante de Pascal Paoli (1775-1796) i. Un ‘peuple presque neuf’, ou les Français redécouvrent les Corses ii. Des amis de circonstance: Sir Gilbert Elliot et son état-major (1794-1796) iii. Le paysage et les hommes vus par les personnalités du royaume anglo-corse iv. La constitution du royaume anglo-corse, et Pascal Paoli, vus par Sir Gilbert Elliot et les personnalités de ce régime Conclusion générale Bibliographie Index

We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating: a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis; an early and late p38 MAPK activation; an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.

Recent studies have shown that copy number variants (CNVs) are important sources of variability of mammalian genomes. We applied a cross species array comparative genome hybridization (aCGH) experiment using as reference the cattle genome to investigate, for the first time, variability in the sheep and goat genomes derived from copy number variation and identified 431 and 358 CNVs, respectively. A comparison of these results to those obtained in other mammals for similar experiments is reported. The identified CNVs could be important in determining phenotypic and production differences between and within breeds. Further studies will be carried out to evaluate the identified CNVs from both functional and evolutionary points of view.

A case of primary infection by human herpesvirus 6 (HHV-6) variant A in a 54-year-old woman, which occurred at the same time as the onset of encephalomyelitis, is reported. The correlation between the two events is discussed. It is speculated that, during the early phase of the infection, the HHV-6 spread to the central nervous system and triggered a pathogenic process that initially developed without symptoms. When the neurological disorders appeared, HHV-6 had already established a latent state: only the virus carried by infected blood cells was detected in the cerebrospinal fluid.

Positive results were attained when human peripheral blood lymphocytes (PBLs) were investigated for their ability to propagate a transmissible cytotoxic activity (TCA) isolated on VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. In consideration of this finding it can be assumed that "in vivo" blood lymphocytes contributed to give rise to the TCA detected "in vitro" in the CSF inoculum.

Twenty five cases of meningitis occurred in urban areas surrounding a city (Modena) in Northern Italy, in the period May-July 1999. When the patients were admitted to the Infectious Diseases Division of the University of Modena and Reggio Emilia Hospital and studied by virological and serological methods, the meningitis proved to have an enteroviral origin and enterovirus ECHO 4 type was responsible for all cases of illness. An epidemiological characteristic of the enteroviral meninigitis outbreak was the adult age in 23 out of the 25 patients (mean age 24.50 +/- 7.84 years). The monthly distribution of the aseptic meningitis cases was the following: five cases occurred in May, 13 in June and seven in July. The origin of the spread of the virus infection and the reason for its sudden end remained unknown. The unusual drop in temperature which occurred in the geographic area involved in the aseptic meningitis outbreak at the beginning of August could have interfered with the slowdown in virus circulation.

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in several cerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusual infectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such an activity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showed a blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection, two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). When neutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein( s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samples disclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reported in previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymes Mn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathway among the causes of the DNA damage induced by the cytotoxic transmissible agent under study.

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172TA, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.

Massese is an Italian sheep breed, with black or grey coat colour, mainly reared in the Tuscany and Emilia Romagna regions. Recently, the emerging interests in this breed have resulted in the production of Pecorino cheese obtained with only Massese milk. In order to be profitable, this marketing link between Massese breed and its products should be defended against fraudsters who could include milk of other sheep breeds or cow milk in Massese labelled productions. To identify the genetic factors affecting coat colour in sheep, we have recently analysed the melanocortin 1 receptor (MC1R) gene and identified several single nucleotide polymorphisms (SNPs). In this work, as a first step to set up a DNA based protocol for authentication of Massese dairy products, we further investigated the presence and distribution of one of these SNPs (c.-31G>A) in 143 Massese sheep and in another 13 sheep breeds (for a total of 351 animals). The Massese breed was fixed for allele c.-31A, whereas in all other breeds allele c.-31 G was the most frequent or with frequency of 0·50. At the same nucleotide position the cattleMC1Rgene carries the G nucleotide. Using these data we developed a method to detect adulterating milk (from other sheep breeds or from cow) in Massese dairy products based on the analysis of the c.-31G>A SNP. We first tested the sensitivity of the protocol and then applied it to analyse DNA extracted from ricotta and Pecorino cheese obtained with only Massese milk or obtained with unrestricted sheep and cattle milk. To our knowledge, this system represents the first one that can be used for breed authentication of a sheep production and that, at the same time, can reveal frauds derived from the admixture of milk of an unreported species.

article i nfo We carried out a cross species cattle-sheep array comparative genome hybridization experiment to identify copy number variations (CNVs) in the sheep genome analysing ewes of Italian dairy or dual-purpose breeds (Bagnolese, Comisana, Laticauda, Massese, Sarda, and Valle del Belice) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. We identified 135 CNV regions (CNVRs; 24 reported in more than one animal) covering ~10.5 Mb of the virtual sheep genome referred to the bovine genome (0.398%) with a mean and a median equal to 77.6 and 55.9 kb, respectively. A comparative analysis between the identified sheep CNVRs and those reported in cattle and goat genomes indicated that overlaps between sheep and both other species CNVRs are highly significant (Pb0.0001), suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Many sheep CNVRs include genes with important biological functions. Further studies are needed to evaluate their functional relevance.

Sequence of the melanocortin 1 receptor (MC1R) gene (the Extension locus) was obtained from a panel of 73 animals belonging to 9 Italian sheep breeds or populations (Appenninica, Bergamasca, Comisana, Cornigliese-like, Delle Langhe, Massese, Merinizzata Italiana, Sarda and Valle del Belice) with different coat colours. Evaluation of the identified polymorphisms on this phenotype was reported with in silico predictions and comparative approaches within and across breeds and across species. Five novel single nucleotide polymorphisms (SNPs), organized in three haplotypes, were detected. Another haplotype, including the two missense mutations already described for the ED allele, was identified in few Massese sheep. One SNP (c.199C > T) caused a predicted amino acid substitution (p.R67C) in a highly conserved position of the first intracellular loop of the MC1R protein. The same substitution causes recessive pheomelanism in other species. We propose that the p.67C allele represents the recessive e allele at the ovine Extension series that was, so far, not completely recognized in sheep by classical genetic studies. This polymorphism was analysed in a total of 388 sheep of the 9 investigated breeds. The p.67C allele was identified only in the Valle del Belice breed (allele frequency of 21.3% in 176 analysed animals of this breed) in which the presence of epistatic white-determining loci might mask, at least in part, its effects. Confirming the effect of this novel allele on coat colour will lead to new perspectives on the composition of specialized coloured sheep lines.

Mammalian coat colour is mainly determined by the distribution of two different types of melanins: pheomelanin (red/yellow pigments) and eumelanin (black pigments). Their synthesis is regulated by the melanocortin 1 receptor (MC1R/Extension locus) that binds the α-melanocyte-stimulating hormone (α-MSH) and the agouti signalling protein (ASIP, coded by the Agouti locus). In mammals, several studies have reported that loss-of-function mutations in MC1R lead to red/yellow pigmentation, while gain-of-function mutations lead to black/dark colours. Mutations at the Agouti locus exert, in general, epistatic interactions on the Extension locus. In goats, classical genetic studies have indicated that variations at the Agouti locus may be the main source of colour variability within and between breeds, while the effect of the Extension locus on this phenotypic trait has been only partially deduced. In order to better understand the role of the Extension locus on coat colour in this species, here we analysed...

Background The goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals. Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector&reg, (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector&reg, profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Le fonds documentaire qui est à la source de cet ouvrage est conservé dans le Fondazione Maria Cosway à Lodi, en Italie. Il comprend essentiellement un corpus de cent vingt-trois lettres et billets datant de 1782 à 1803, qui constitue la correspondance active du chef corse Pascal Paoli (1725-1807) à Maria Cosway (1760-1838), l’épouse du célèbre peintre miniaturiste Richard Cosway. Chaque document, dont l’original est en italien, est retranscrit, traduit et annoté; l’édition comporte en outre une chronologie historique, une chronologie des lettres et un index. Cette correspondance éclaire sous un jour nouveau la biographie de Pascal Paoli au temps de son séjour en Angleterre, période qui constituait une lacune importante dans ce domaine. Elle précise la nature des liens entre le chef corse et la famille Bonaparte. Envers sa correspondante, l’auteur se révèle malicieux, spirituel, affectueusement ironique, moralisateur, ferme sur le décorum qu’il croit devoir maintenir, mais aussi traversé parfois par le doute, la tristesse et le découragement, voire l’angoisse métaphysique. Le portrait moral qui en ressort est celui d’un personnage amateur de littérature, de peinture et de musique, plus sensible, plus vivant, moins figé que l’image littéraire héroïsée et ‘monumentale’ du ‘champion de la liberté’ que James Boswell a laissé à la postérité dans son Account of Corsica and Memoirs of Pascal Paoli. On perçoit aussi la riche et attachante personnalité de la destinaire de ces lettres. Le seul fait que Maria Cosway ait eu le souci de conserver ces documents témoigne moins de sa vanité que de son intelligence. Un autre intérêt, et non des moindres, est la toile de fond, esquissée par les nombreuses allusions à la pléiade de célébrités du monde des lettres et des arts que Paoli côtoyait. Cette évocation illustre à quel point Londres était alors un ardent et brillant foyer de culture. Liste des illustrations Avant-propos Remerciements Abréviations et citations Chronologie biographique Chronologie de la correspondance Introduction I. Lettres datées de Pascal Paoli à Maria Cosway II. Lettres non datées III. Autres documents Liste des ouvrages cités Index

The agouti locus encodes the agouti signalling protein (ASIP) which is involved in determining the switch from eumelanin to pheomelanin synthesis in melanocytes. In the domestic rabbit (Oryctolagus cuniculus) early studies indicated three alleles at this locus: A, light-bellied agouti (wild type); at, black and tan; a, black nonagouti. We characterized the rabbit ASIP gene and identified the causative mutation (an insertion in exon 2) of the black nonagouti allele whose frequency was evaluated in 31 breeds. Phylogenetic analysis of ASIP sequences from Oryctolagus and 9 other species of the family Leporidae placed Oryctolagus as sister species to Pentalagus and Bunolagus. Transcription analysis in wild type agouti rabbits revealed the presence of two major transcripts with different 5′-untranslated regions having ventral or dorsal skin specific expression. ASIP gene transcripts were also detected in all examined rabbit tissues distinguishing the rabbit expression pattern from what was observed in wild type mice.

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship. J. Cell. Physiol. 220: 553–561, 2009. © 2009 Wiley-Liss, Inc.

A proteomic-based search for novel substrates of Akt was undertaken in C2C12 murine muscle cells. Our data demonstrate that Akt isoform 2 phosphorylates Ankrd2 at Serine 99 in response to H2O2 stimuli, regulating muscle differentiation rate., Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

Background In the domestic rabbit (Oryctolagus cuniculus), classical genetic studies have identified five alleles at the Extension locus: ED (dominant black), ES (steel, weaker version of ED), E (wild type, normal extension of black), eJ(Japanese brindling, mosaic distribution of black and yellow) and e (non-extension of black, yellow/red with white belly). Sequencing almost the complete coding sequence (CDS) of the rabbit MC1R gene, we recently identified two in-frame deletions associated with dominant black (c.280_285del6; alleles ED or ES) and recessive red (c.304_333del30; allele e) coat colours. It remained to characterize the eJallele whose phenotypic effect is similar to the Orange and Sex-linked yellow loci of cat and Syrian hamster. Results We sequenced the whole CDS in 25 rabbits of different coat colours including 10 Japanese and 10 Rhinelander (tricolour) rabbits and identified another 6 bp-in frame deletion flanked by a G > A transition in 5' (c.[124G>A;125_130del6]) that was present in all animals with Japanese brindling coat colour and pattern. These mutations eliminate two amino acids in the first transmembrane domain and, in addition, cause an amino acid substitution at position 44 of the wild type sequence. Genotyping 371 rabbits of 31 breeds with different coat colour this allele (eJ) was present in homozygous state in Japanese, Rhinelander and Dutch tricolour rabbits only (except one albino rabbit). Rabbits with eJ/eJ genotype were non fixed at the non-agouti mutation we previously identified in the ASIP gene. Segregation in F1 and F2 families confirmed the order of dominance already determined by classical genetic experiments with a possible dose effect evident comparing eJ/eJ and eJ/e animals. MC1R mRNA was expressed in black hair skin regions only. Conclusions The c.[124A;125_130del6] allele may be responsible for a MC1R variant determining eumelanin production in the black areas. However, the mechanism determining the presence of both red and black hairs in the same animal seems more complex. Expression analyses of the c.[124A;125_130del6] allele suggest that MC1R transcription may be regulated epigenetically in rabbits with the Japanese brindling phenotype. Further studies are needed to clarify this issue.

The objective of this study was to evaluate the effect of postmortem times on the quality of porcine skeletal muscle total RNA in order to consider the possibility to use postmortem material for gene expression analysis. Samples of Musculus semimembranosus were collected at 20 min, 2 h, 6 h, 24 h and 48 h postmortem from the left legs of four commercial heavy pigs. Total RNA was analysed by agarose gel electrophoresis stained with ethidium bromide and by microfluidic capillary electrophoresis on an Agilent 2100 Bioanalyzer instrument obtaining 28S:18S rRNA peak ratios and RIN values. The average RIN values of the analysed samples were 7.45 ± 0.13, 7.43 ± 0.15, 7.45 ± 0.10, 7.33 ± 0.15 and 3.95 ± 0.58 for the same postmortem times, respectively, indicating that RNA degradation was present at 48 h postmortem . In a similar experiment, carried out by other authors on beef cattle muscle total RNA extracted at different postmortem times, RNA was stable up to 8 days after death as indicated by intact 28S and 18S rRNA bands. Thus, differences among species or other environmental factors might affect the level of RNA degradation. In the porcine postmortem samples, qualitative assessment of GAPDH transcripts by PCR amplification of different cDNA fragments indicated that postmortem stages did not affect the possibility of analysing this housekeeping gene. Thus, postmortem porcine skeletal muscle can be an useful tissue to obtain gene expression based information.