Antonella Palomba

A comparison of conservative DNA extraction methods from fins and scales of freshwater fish: A useful tool for conservation genetics

Key words: brown trout, conservative DNA extraction, fish fin clip, fish scales, northern pikeNon-destructive protocols for DNA isolationfrom fresh or preserved specimens are fundamen-tal to study endangered or elusive species, breedersor samples sibship and specimens derived frompublic or private collections (Nielsen et al. 1999;Wasko et al. 2003; Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yieldand poor quality DNA, particular laboratory careand standardizations were needed to allow repro-ducible results. We compared six DNA extractionprocedures from conservative samples: three arecommercial kits [Wizard Genomic DNA Purifi-cation Kit (WGDPK) (Promega); Wizard Mag-netic DNA Purification System for Food(WMDPF) (Promega); NucleoSpin Food (NSF)(Macheray–Nagel)] and three are largelyemployed methodologies [Trizol (Life Technolo-gies); Chelex (Sigma–Aldrich); C-TAB]. For eachmethod, the standard protocol (reported on datasheets or the first published one) and severalvariations were tested varying sample storage andpre-lysis conditions, homogenization procedures,buffer solutions and concentrations, incubationsand resuspension times and temperatures. DNAwas extracted from 200 northern pikes (Esoxlucius L.) and 100 brown trout (Salmo trutta farioL.) (Table 1) from small (£10 mg) fin pieces and5–10 scales stored dried or in absolute alcoholboth at )20 C and at room temperature. DNAextraction was carried out on fresh materials andrepeated within a period of three years (Table 1),and from 34 years old dried scales culled from amuseum collection. Furthermore, DNA wasextracted from liver and muscle of both species,from individuals that had naturally died and usedas controls. Spectrophotometric readings (260 and280 nm) and densitometric evaluations (Image J)of DNAs obtained through different methodolo-gies and protocols (conservative versus controlDNA) were analyzed by means of ANOVA andt-test. DNA suitability was assayed through PCR–RFLP and microsatellite analyses (Lucentini et al.(2006) (Table 2). Microsatellites were run onABI377 DNA sequencer whereas PCR–RFLPswere assayed on 2.5% agarose electrophoresis fortwo mtDNA NADH coding regions (ND-1 ND5/6) (Cronin et al. 1993). DNA stability andamplificability was controlled every three months,for three years, in parallel with control DNA. Toevaluate genotyping errors, null alleles presenceand allelic dropout, all the experiments were rep-licated and statistically treated as suggested(Hoffman and Amos 2005; Roon et al. 2005)through MICRO-CHECKER 2.2.3 (Vanoosterhout et al. 2004). Deviations from theHardy–Weinberg equilibrium were tested byArlequin 2000 and Genepop.Our results indicate that the quality and quan-tity of DNA extracted from fin clips and scalesvaried according to extraction methods (Table 2)and storage conditions. Alcohol preservation at)20 C is fundamental for fin specimens, althoughdried scales conservation at room temperatureallowed good DNA extraction. One percent

Genetic characterization of a putative indigenous brown trout (Salmo trutta fario) population in a secondary stream of the Nera River Basin (Central Italy) assessed by means of three molecular markers

Genetic diversity was analysed in a putative autochthonous brown trout (Salmo trutta fario L.) population (Monterivoso Stream, Tyrrhenian Apennine Slope) by means of seven microsatellite loci and PCR‐RFLP of two mitochondrial (ND1 and ND5/6) and one nuclear (LDH‐C1*) locus. Monterivoso data were compared to those obtained analysing populations of the same basin (Nera River) and of the Po basin; Irish populations were used as a source of Atlantic strain brown trout. Haplotypes distributions, heterozygosity, F‐statistic and UPGMA analyses indicated a genetic diversification between these populations, suggesting a widespread alteration of the genetic structure due to repeated stocking with allochthonous material, mainly of Atlantic origin, that has partially polluted the Monterivoso population. This population appeared to be constituted of Mediterranean strain samples and might represent a residue of an indigenous pool: it shows high specificity characteristics and it is genetically separate from the others observed and from populations of the Adriatic slope of the Apennines. This population should be employed in managing and breeding programmes finalised to an eco‐sustainable restocking of brown trout in the Nera River Basin, with periodic monitoring by genetic analyses.

Exhaled nitric oxide in children with allergic rhinitis: A potential biomarker of asthma development

Pearce N, Foliaki S, Wong G. Birthweight and the risk of atopic diseases: the ISAAC Phase III study. Pediatr Allergy Immunol 2014: 25: 264–70. 5. Briana D, Malamitsi-Puchner A. Small for gestacional age birth weight: impact on lung structure and function. Paediatr Respir Rev 2013: 4: 256–62. 6. Gluckman P, Hanson M, Cooper C, Thornburg K. Effect of in utero and earlylife conditions on adult health and disease. N Engl J Med 2008: 359: 61–73. 7. Duijts L. Fetal and infant origins of asthma. Eur J Epidemiol 2012: 27: 5–14. 8. Murphy V, Namazy J, Powell H, et al. A meta-analysis of adverse perinatal outcomes in women with asthma. BJOG 2011: 118: 1314–23. 9. Wandalsen G, Chong-Neto H, Souza F, Sol e D, Bacharier L. Early weight gain and the development of asthma and atopy in children. Curr Opin Allergy Clin Immunol 2014: 14: 126–30.

Muscle actin isoforms are differentially expressed in human satellite cells isolated from donors of different ages.

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied α‐smooth muscle (αSMA) and α‐sarcomeric muscle (αSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5‐day‐old newborn, a 34‐year‐old young adult and a 71‐year‐old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time‐course expression of αSRA and αSMA were studied with both immunocytochemistry and western blotting procedures. Although αSMA was never detected in whole skeletal muscle, both αSMA and αSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that αSRA was gradually up‐regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re‐expressed αSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.

Genetic differentiation among populations of Baetis rhodani (Ephemeroptera, Baetidae) in three Italian streams

Abstract Seven microsatellite loci were recently described for the mayfly Baetis rhodani, but, at present, no data are available on the population structure of this species using these loci. We used four of these microsatellite loci for a preliminary analysis of the genetic differentiation of the mayfly B. rhodani in six populations from three Italian streams belonging to the same or different, but geographically distant, drainage basins. From these data, genetic polymorphism was observed between the sampled populations and clear differences were identified between pairs of populations; however, no specific population pattern was observed. The high FST values suggest a limited dispersal of the species across the sampling sites. On the whole, the results obtained with microsatel‐lite loci, characterized by marked heterozygosity deficiency, confirm the hypothesis of the patchy recruitment from a small number of matings and limited in‐stream dispersal, as previously pointed out for some other baetid mayflies by genetic analyses using al‐lozymes and mtDNA. This is the first contribution to a knowledge of the genetic structure of Italian Baetis rhodani populations.

PCR-RFLP approaches to easily identify Pleuronectes platessa from other flatfishes: a rapid and efficient tool to control label information

European plaice (Pleuronectes platessa) is quite frequently substituted with other flatfish, especially with Yellow fin sole (Limanda aspera), which differs not only in meat quality but also particularly in its origin and manipulation chain. We propose an integrated approach of laboratory and in silico mtDNA PCR-RFLP procedures generating a set of restriction patterns easily resolvable in agarose gel, able to discriminate with certainty P. platessa from other 20 flatfish species. The herein proposed procedure is an economical and valid tool in detecting mislabelled seafoods.

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.