Leccese sheep breed from Apulia: evidence of genetic substructuring from STR loci

Leccese is an originally triple-purpose sheep breed from Southern Italy. Due to major changes in rural society and livestock production, the breed has experienced in the last decades a drastic population size reduction. Nowadays roughly one thousand animals are reared in about ten farms located in Apulia. In order to evaluate the genetic variability within the Leccese breed, a total of 97 animals, sampled from five different farms in the provinces of Bari, Taranto, Brindisi, and Lecce were genotyped at 19 microsatellite loci belonging to the ISAG-FAO panel for Domestic Animal Diversity. Samples were chosen according to genealogical records in order to minimize relatedness among animals. Although being a local bottlenecked breed, the Leccese sample displayed a valuable number of alleles (187 over 19 loci, with a minimum of 4 in BM1824 and OarFCB193 and a maximum of 16 in MAF70 and MCM140). A high proportion of private alleles was observed for each farm (7.7 alleles on average). In addition, a high level of linkage disequilibrium was observed in the total sample, also among non syntenic locus pairs, suggesting the presence of population sub-structuring. To test the hypothesis of a genetic partitioning at the farm hierarchical level we first calculated the pair-wise FST between the different farms, which highlighted significant (P<0.001) though moderate values (0.055 to 0.065). Then, we performed both a likelihood-based and a Bayesian population assignment test, respectively implemented in the Arlequin and Structure software packages, in order to verify the degree of differentiation between the five farms. A correct allocation was obtained for 96.9% of animals by using the Arlequin software and 92.8% when using Structure, thus highlighting a clear genetic differentiation at the farm level, counterbalanced by a marked within-herd genetic similarity. These results suggest that reproductive isolation and/or different selection strategies across farms contributed to the observed pattern of genetic sub-structuring; this should be seriously taken into consideration, due to the critical implications for the breed conservation

Analisi della variabilità genetica in una popolazione ovina di razza massese e studio di associazione con parametri di qualità del latte

RIASSUNTO Il presente lavoro ha come obiettivo la ricerca di associazioni tra marcatori microsatellite e la composizione chimica del latte. È stato condotto uno studio su 68 pecore di razza Massese provenienti da un allevamento toscano. A partire da latte fresco, sono stati valutati: composizione chimica standard, contenuto in caseina e frazioni caseiniche, proteine seriche, pH e parametri reologici. I capi sono stati sottoposti a genotipizzazione impiegando 17 marcatori microsatellite; sono, quindi, stati calcolati i valori di similarità genetica tra individui ed alcuni parametri genetici classici. Il numero medio di alleli per locus è risultato pari a 7,18 e l’eterozigosità osservata presentava variazioni tra 0,403 e 0,867 (media 0,677). La similarità genetica tra individui era 0,460. Cinque marcatori (BM8124, CSN3, BM1258, BMS468 e TGLA387) hanno mostrato una deviazione significativa dalle proporzioni di Hardy- Weinberg. Lo studio ha messo in evidenza alcuni microsatelliti con alleli significativamente associati a caratteri di composizione del latte (P<0,01). In particolare, la significatività più alta (P<0,001) è stata osservata per l’associazione dell’allele 2 del marcatore OIFNG con la a-lattoalbumina, dell’allele 10 del marcatore BL4 con il livello di immunoglobuline, dell’allele 2 del marcatore BMC1009 con il contenuto di grasso e dell’allele 9 del marcatore ILSTS42 con il parametro a30. Sono necessari ulteriori approfondimenti al fine di convalidare i risultati preliminari ottenuti nel presente lavoro e si rende necessaria l’estensione dello studio ad un numero maggiore di soggetti e la tipizzazione di un numero maggiore di loci, principalmente sui cromosomi 3 e 20 dove mappano i marcatori risultati più interessanti nella presente analisi. SUMMARY Current research aims to establish statistical associations between DNA microsatellites and milk chemical composition. Atrial was carried out on 68 Massese ewes reared in a farm of the Tuscany. The following parameters were evaluated on fresh milk: standard chemical composition, casein and its fractions, whey proteins, pH and rheological parameters. Animals were genotyped at 17 microsatellite loci. Genetic similarities among individuals and classical genetic parameters were evaluated. For each locus, average values of considered parameters were calculated in all the subjects carrying a given allele; these were compared statistically with the average values of subjects not carrying the allele, and the significance of the difference was estimated. The average number of alleles per locus resulted 7.18 and the observed heterozygosity ranged from 0.403 to 0.867 (0.677 medium value). The genetic similarity among individuals was 0.460. Five markers pointed out a significant deviation from the Hardy-Weinberg proportions (BM8124, CSN3, BM1258, BMS468 and TGLA387). The study revealed several microsatellites with alleles significantly linked to milk composition traits (P<0.01). In particular the highest significance (P<0.001) has been found for the allele 2 of OIFNG marker with a-lactoalbumin, for the allele 10 of the BL4 marker with immunoglobulins, for the allele 2 of BMC1009 with fat, and for allele 9 of ILSTS42 with a30. Further analyses are needed to validate these preliminary results, in particular increasing the number of subjects and of typed loci above all on the chromosomes 3 and 20 where the more interesting markers map

Genetic variability of three local cattle breeds (Calvana, Pontremolese, Garfagnina) by STR analysis

AbstractThe dramatic size contraction of local cattle breeds due to replacement with cosmopolite improved breeds highlights the need for native genetic resources conservation. In 1985, the Anagraphic Register of local cattle breeds and small-size ethnic groups was established by the Italian Ministry of Agriculture and Forestry. Calvana, Pontremolese and Garfagnina are among the included breeds. They are all native from Tuscany. Present breeding area covers the provinces of Firenze, Prato, Pistoia and Siena for the Calvana breed (around 280 heads), while it is restricted to the province of Lucca for both Garfagnina (around 180 heads) and Pontremolese. This latter breed consists, nowadays, of less than 40 heads, while being around 15000 in 1940s.The characterization of the genetic structure and variability via molecular markers could provide useful information for breed management and conservation. In the present study, a total of 149 animals, evenly distributed among the three breeds, were genetically char..

Haplotype recostruction from unphased genotype data at the bovine PRKAG3 gene

Haplotype analysis has became an area of intense research, both for population genetics studies and for molecular dissection of complex phenotypes. Haplotypes provide increased informativeness with respect to single nucleotide polymorphisms and allow to condense information on genomic variation, with a sensible gain of power in association studies. Direct haplotyping via molecular analysis provides more exact information per individual, but is much more expensive and labour-intensive than indirectly inferring haplotypes from genotypes. Several methods have been proposed so far to indirectly reconstruct haplotypes from unphased genotypes. In the present study, we adopted four different approaches (implemented in the computer programs ARLEQUIN, HELIXTREE, HAP and PHASE) to infer phase information from genotypic data on 197 subjects at 14 polymorphic sites of the bovine PRKAG3 gene. In the whole, twelve different haplotypes had been inferred by all the four different methods, although at slightly varying frequencies. Other three haplotypes were inferred by at least two different approaches and some haplotypes were unique to a single method. No dramatic differences among the four selected approaches were observed for the considered genomic target. This is probably due to the linkage-disequilibrium structure of the analysed region and to the moderate amount of missing genotype data

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Centrin 2: a novel marker of mature and neoplastic human astrocytes

As microtubule organizing centers, centrosomes play a pivotal role in cell division as well as in neurodevelopment and neuronal maturation. Among centrosomal proteins, centrin-2 (CETN2) contributes also to DNA repair mechanisms which are fundamental to prevent genomic instability during neural stem cell pool expansion. Nevertheless, the expression profile of CETN2 in human neural stem cells and their progeny is currently unknown. To address this question, we interrogated a platform of human neuroepithelial stem (NES) cells derived from post-mortem developing brain or established from pluripotent cells, and demonstrated that while CETN2 retains its centrosomal location in proliferating NES cells, its expression pattern changes upon differentiation. In particular, we found that CETN2 is selectively expressed in mature astrocytes with a broad cytoplasmic distribution. We then extended our findings on human autoptic nervous tissue samples. We investigated CETN2 distribution in diverse anatomical areas along the rostro-caudal neuraxis and pointed out a peculiar topography of CETN2-labelled astrocytes in humans which was not appreciable in murine tissues, where CETN2 was mostly confined to ependymal cells. As prototypical condition with glial overproliferation, we also explored CETN2 expression in glioblastoma multiforme, reporting a focal concentration of CETN2 in neoplastic astrocytes. This study expands CETN2 localization beyond centrosomes and reveals a unique expression pattern which makes it eligible as a novel astrocytic molecular marker, thus opening new roads to glial biology and human neural conditions

Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons

Background: Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. Methods: We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection we analysed layer II-III pyramidal neurons molecular profile and with Local Field Potentials (LFP) recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. Results: We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP-25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (i.e., AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signalling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in two mouse models of tumor-bearing mice. Conclusions: These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life

Effects of short-term hyperoxia on erythropoietin levels and microcirculation in critically Ill patients: a prospective observational pilot study

BACKGROUND: The normobaric oxygen paradox states that a short exposure to normobaric hyperoxia followed by rapid return to normoxia creates a condition of 'relative hypoxia' which stimulates erythropoietin (EPO) production. Alterations in glutathione and reactive oxygen species (ROS) may be involved in this process. We tested the effects of short-term hyperoxia on EPO levels and the microcirculation in critically ill patients.METHODS: In this prospective, observational study, 20 hemodynamically stable, mechanically ventilated patients with inspired oxygen concentration (FiO2) \ue2\u89\ua40.5 and PaO2/FiO2\ue2\u80\u89\ue2\u89\ua5\ue2\u80\u89200\uc2\ua0mmHg underwent a 2-hour exposure to hyperoxia (FiO2 1.0). A further 20 patients acted as controls. Serum EPO was measured at baseline, 24\uc2\ua0h and 48\uc2\ua0h. Serum glutathione (antioxidant) and ROS levels were assessed at baseline (t0), after 2\uc2\ua0h of hyperoxia (t1) and 2\uc2\ua0h after returning to their baseline FiO2 (t2). The microvascular response to hyperoxia was assessed using sublingual sidestream dark field videomicroscopy and thenar near-infrared spectroscopy with a vascular occlusion test.RESULTS: EPO increased within 48\uc2\ua0h in patients exposed to hyperoxia from 16.1 [7.4-20.2] to 22.9 [14.1-37.2] IU/L (p\ue2\u80\u89=\ue2\u80\u890.022). Serum ROS transiently increased at t1, and glutathione increased at t2. Early reductions in microvascular density and perfusion were seen during hyperoxia (perfused small vessel density: 85% [95% confidence interval 79-90] of baseline). The response after 2\uc2\ua0h of hyperoxia exposure was heterogeneous. Microvascular perfusion/density normalized upon returning to baseline FiO2.CONCLUSIONS: A two-hour exposure to hyperoxia in critically ill patients was associated with a slight increase in EPO levels within 48\uc2\ua0h. Adequately controlled studies are needed to confirm the effect of short-term hyperoxia on erythropoiesis.TRIAL REGISTRATION: ClinicalTrials.gov ( www.clinicaltrials.gov ), NCT02481843 , registered 15th June 2015, retrospectively registered

Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.

Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.This work was partially supported by the Italian Ministry of Health (Ministero della Salute, Ricerca Corrente 245)S

The SUCCESSO-TERRA Project: a Lesson of Sustainability from the Terramare Culture, Middle Bronze Age of the Po Plain (Northern Italy)

This backstory article deals with the SUCCESSO-TERRA Project (2017–2020), an interdisciplinary research program aiming at reconstructing the land-use transformations that occurred during the development of the Terramare culture in the southern-central Po Plain of Northern Italy. Topics include climate-environment changes, human impact and exploitation of natural resources that are interconnected topics in human ecology and environmental sciences. These topics can only be understood in a long-term perspective integrating archaeology, geology, botany and other sciences. The text includes the theoretical basis, the research strategy and the main methodological approaches given by geoarchaeology and palynology, the two research sides constituting the partnership of the project