We report the development of a system to feed pulsed magnetic stress to biological samples. The device is based on a RLC circuit that transforms the energy stored in a high voltage capacitor into a magnetic field inside a coil. The field has been characterized and we found that charging the capacitor with 24 kV results in a peak field of 0.4 T. In order to test its effect, we applied such a stress to the Drosophila melanogaster model and we examined its bio-effects. We analysed, in the germ cells, the effects on the control of specific DNA repetitive sequences that are activated after different environmental stresses. The deregulation of these sequences causes genomic instability and chromosomes breaks leading to sterility. The magnetic field treatment did not produce effects on repetitive sequences in the germ cells of Drosophila. Hence, this field doesn't produce deleterious effects linked to repetitive sequences derepression.

The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from high-density lipoproteins (HDL) to triglyceride (TG)-rich lipoproteins. A consistent number of investigations has suggested an association between the TaqIB polymorphism of the CETP gene, plasma HDL-C levels and the risk of cardiovascular disease, but the results are controversial. The aim of this study was to determine if the TaqIB polymorphism might be related to the presence of atrial fibrillation (AF). We conducted a case-control study, enrolling 109 Caucasian unrelated patients coming from Salento (Southern Italy) with documented AF and 109 controls selected from the same ward. The CETP TaqIB genotypes were determined by RFLP-PCR. The subjects with the B2B2 genotype seem to be more susceptible to AF development (OR=2.28, 95% CI 1.06-4.89, p=0.032). The AF incidence is higher if we consider only the female subgroup (OR=5.14, 95% CI 1.57-16.82, p=0.0061). In the AF female subgroup the B2B2 patients had a statistically significant decrease of HDL-C levels (1.50 ± 0.35 vs 2.07 ± 0.42; p=0.012) and statistically higher TG levels (1.34 ± 0.46 vs 0.77 ± 0.14; p=0.027) and TG/HDL-C ratio (2.14 ± 0.80 vs 0.88 ± 0.23; p=0.007) when compared to B2B2 female control subjects. When we analyzed the linkage between the TaqIB polymorphism and the promoter variant (-629C/A), we found that 100% of the B2 alleles of the TaqIB polymorphism were associated with the A alleles of the -629 promoter polymorphism in our subjects.This study suggests that in post-menopausal women atrial fibrillation could be promoted by the association of CETP B2B2/AA genotype with higher triglycerides values.

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

The pseudocapsule (PC) of the uterine leiomyoma (UL) is an anatomic entity that surrounds the myoma separating it from the myometrium (UM). Although a number of microarray experiments have identified differences in gene expression profile in the UL when compared with the UM, there is a lack of systematic studies on the PC. In this study, quantitative RTPCR analysis was performed on 18 matched PC, UL and UM specimens and results showed that the PC displays a specific gene expression profile. The low expression level of insulin-like growth factor (IGF-2), a fibroid specific marker, that we found in the PC and the UM when compared with the UL, clearly indicates that the PC is in structural continuity with the UM. However, the significant increase in endoglin expression level in PC with respect to the UL and UM indicates that an active neoangiogenesis is present in PC. Conversely, other angiogenic factors such as von Willebrand factor (vWF) and vascular endothelial growth factor A (VEGF-A) seem to have little influence on the PC angiogenesis. Because the endoglin is preferentially expressed in proliferating endothelial cells, whereas the vWF and VEGF-A are preferentially expressed in preexisting endothelial cells, our idea is that the angiogenic activity in the PC is linked to wound healing. The angiogenic activity is also sustained by intermediate expression level of cystein-rich angiogenesis inducer 61, connective tissue growth factor and collagen 42 genes all involved in the neoangiogenesis, that we detected in the PC. Taken together our data demonstrate that the specific expression pattern observed in the PC could be the response of the uterine walls smooth cells to the tension imposed by the tumor. As a consequence, a neovascular structure is generated involving regenerative processes. For these reasons, we suggest that the laparoscopic intracapsular myomectomy (LIM), a new surgical technique that preserves the PC during the UL removal, should always be preferred, to favor a faster and proper uterine healing.

The canalization concept, first introduced by Waddington1, describes the resistance of a developmental process to phenotypic variation regardless of genetic and environmental perturbations, thanks to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and then can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism itself could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways2-5, seem to have identified it as a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability6,7. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. There has been a great interest in this proposal of a concrete mechanism underlying canalization. We would like to offer an additional, perhaps alternative, explanation for these observations. We show that, in Drosophila, functional alterations of Hsp90 affect the piRNA silencing mechanism leading to transposon activation and the induction of morphological mutants. This suggests that Hsp90 mutations can actually generate new variation by trasposon-mediated “canonical” mutagenesis.

Pol32 is an accessory subunit of the replicative DNA Polymerase delta and of the translesion Polymerase zeta. Pol32 is involved in DNA replication, recombination and repair. Pol32's participation in high-and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32(rd1) and pol32(rds) in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

Aubergine is an RNA-binding protein of the Piwi clade, functioning in germline in the piRNA pathway that silences transposons and repetitive sequences. Several mutations of this gene exist, but they mostly result in truncated proteins or correspond to mutations that also affect neighboring genes. We have generated complete aubergine knock-out mutants that do not disrupt the neighboring genes. These novel mutants are characterized by PCR and sequencing. Their nature is confirmed by female sterility and by the presence of crystals in testes, common to the aubergine loss of function mutations. These mutants provide novel and more appropriate tools for the study of the piRNA pathway that controls genome stability.

Background: The aim of this study was to determine the occurrence of gluten-sensitivity (GS) in a group of allergic patients and assess the efficacy of a gluten free diet (GFD) on the improvement of the symptomatology in those who were diagnosed with GS. Methods: 262 unrelated allergic patients, with gastrointestinal symptoms of obscure origin were proven for GS condition by biopsy. All patients were also genotyped for the typical celiac DQ2 and DQ8 molecules and investigated for several hematological parameters such as antigliadin and antiendomysial antibodies. Patients displaying mucosal lesions were invited to follow a GFD. Results: 77 of the 262 allergic patients were positive to mucosal lesions, but negative to the antiAGA, antiEMA and to DQ2 and DQ8 molecules. We found, instead, a prevalence of the DQA1*05 allele, whereas anemia of inflammatory origin, represented the predominant complaint in our subjects. The positive patients who, after the GS diagnosis, followed a gluten free diet, exhibited the control of symptoms, the stabilization of the hematological parameters even if allergic manifestations were not abated. Conclusions. A non-celiac gluten-sensitive (NCGSE) commonly occurs in allergic patients. Based on the high prevalence of NCGSE in allergy, it is recommended that biopsy should be part of the routine investigation of allergic disease to offer the benefits of treatment with a gluten free diet to the patients.

Magnetic field effects are diffused among living organisms. They are mainly studied with static or extremely low frequency fields, while scarce information is available for pulsed fields. This work is devoted to the study of the interaction between Drosophila melanogaster, both adults and larvae, and pulsed magnetic fields. We exposed the organisms to a peak field of 0.4 T, lasting for about 2 μ s, within an ad hoc designed copper coil. Adult individuals didn't present any deregulation of repetitive sequences in the germ line of Drosophila. Instead, we noticed a marked magnetic field effect in larvae. Polytene chromosomes coming from treated individuals showed the presence of heat shock puffs; the same organisms revealed also an upregulation of the genes encoding for the Hsp70 protein. These observations suggest that the larvae underwent an oxidative stress caused by the modulation of free radicals' yield induced by the magnetic field through a radical pair mechanism.

The Stellate-made crystals formation in spermatocytes is the phenotypic manifestation of a disrupted crystal-Stellate interaction in testes of Drosophila melanogaster. Stellate silencing is achieved by the piRNA pathway, but many features still remain unknown. Here we outline the important role of the crystal-Stellate modifiers. These have shed light on the piRNA pathways that defend genome integrity against transposons and other repetitive elements in the gonads. In particular, we illustrate the finding that HSP90 participates in the molecular pathways of piRNA production. This observation has relevance for the mechanisms underlying the evolutionary canalization process.

RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.