Small RNAs in eucaryotes: new clues for amplifying microRNA benefits

miRNAs, the smallest nucleotide molecules able to regulate gene expression at post transcriptional level, are found in both animals and plants being involved in fundamental processes for growth and development of living organisms. The number of miRNAs has been hypothesized to increase when some organisms specialized the process of mastication and grinding of food. Further to the vertical transmission, miRNAs can undergo horizontal transmission among different species, in particular between plants and animals. In the last years, an increasing number of studies reported that miRNA passage occurs through feeding, and that in animals, plant miRNAs can survive the gastro intestinal digestion and transferred by blood into host cells, where they can exert their functions modulating gene expression. The present review reports studies on miRNAs during evolution, with particular focus on biogenesis and mechanisms regulating their stability in plants and animals. The different biogenesis and post biogenesis modifications allow to discriminate miRNAs of plant origin from those of animal origin, and make it possible to better clarify the controversial question on whether a possible cross-kingdom miRNA transfer through food does exist. The majority of human medicines and supplements derive from plants and a regular consumption of plant food is suggested for their beneficial effects in the prevention of metabolic diseases, cancers, and dietary related disorders. So far, these beneficial effects have been generally attributed to the content of secondary metabolites, whereas mechanisms regarding other components remain unclear. Therefore, in light of the above reported studies miRNAs could result another component for the medical properties of plants. miRNAs have been mainly studied in mammals characterizing their sequences and molecular targets as available in public databases. The herein presented studies provide evidences that miRNA situation is much more complex than the static situation reported in databases. Indeed, miRNAs may have redundant activities, variable sequences, different methods of biogenesis, and may be differently influenced by external and environmental factors. In-depth knowledge of mechanisms of synthesis, regulation and transfer of plant miRNAs to other species can open new frontiers in the therapy of many human diseases, including cancer

Efficacy of safety catheter devices in the prevention of occupational needlestick injuries: applied research in Liguria Region (Italy)

Health care workers who use or may be exposed to needles are at increased risk of needlestick injuries which can lead to serious infections with bloodborne pathogens. These injuries can be avoided by eliminating the unnecessary use of needles, using safety devices. The present study was aimed at evaluating the impact of a safety-engineered device, with passive fully automatic needlestick protection, on the reduction of needlestick injuries among health care workers. The setting of the study was a network of five public health care institutions situated in a Northern Italian Region. Data about the type of device, the number of employees and the amount of catheter devices used per year were collected through regular meetings with health care workers over a period of five years.The most remarkable result of this study was represented by the huge risk reduction estimated for safety devices. Indeed, the risk of needlestick injuries due to conventional devices was found to be 25 fold higher than that observed for safety devices. However, it is noteworthy that a discernible part of this excess can be explained by the different background amount of devices used. Moreover, the descriptive analysis suggested that individuals with a poor/moderate training level showed a lower risk, albeit not statistically significant, than those with a good/high training.In conclusion, there is a convincing evidence of a causal connection between the introduction of safety devices and reduction in the occurrence of needlestick injuries. This consideration pushes to introduce safety devices into daily clinical practice

2003-07

Background and Objectives. B-cell chronic lymphocytic leukemia (B-CLL) results from the accumulation of monoclonal CD5 + B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous. Clinical studies indicate that CD38 + B-CLL are characterized by a more aggressive clinical course than are CD38 − B-CLL. On the basis of these studies and considering the established correlation between specific chromosome aberrations and the clinical course of B-CLL, it is possible that CD38 + B-CLL cases are also characterized by specific subsets of chromosomal alterations. Design and Methods. Comparative genomic hybridization (CGH) was performed on purified B-cells from peripheral blood of 52 patients with B-CLL in order to detect chromosome imbalance. The immunophenotype of the patients, including CD38 expression, was also determined by flow cytometry. The results of CGH experiments were then compared with CD38 expression. Results. We found a clear correlation between the presence of chromosomal imbalances and CD38 expression: 13/16 CD38 + cases had chromosome imbalances, most of them (12/13) correlated with a poor prognosis. Among the CD38 − B-CLL patients, only 8/36 displayed chromosome imbalances; the only three cases with loss in 13q as a single aberration, considered a good prognostic marker, were in this group. Moreover, we found that cytogenetic alterations were also more complex in the CD38 + B-CLL subset, since 9/10 with two or more aberrations were in the CD38 + group. Interpretation and Conclusions. Collectively, the data reinforce the value of CD38 as a prognostic factor and indicate that genotypic/phenotypic features distinguish B-CLL subsets. Key words: B-CLL, molecular cytogenetics, chromosome aberrations, immunophenotype, CD38. from the progressive accumulation of monoclonal CD5-positive B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous since some patients survive for a long time without therapy, while others progress towards more advanced stages and die despite aggressive treatment. Design and Methods Clinical features of patients Fifty-two patients with B-CLL (32 males and 20 females) were studied. Their characteristics are described in n.a. data not available; *from diagnosis. © F e r r a t a S t o r t i F o u n d a t i o n haematologica/journal of hematology vol. 88 CGH To increase the sensitivity of the CGH, B cells were purified from PBMC by removing monocytes and CD3 + T cells by adherence to plastic surfaces and magnetic beads, respectively. DNA extraction and CGH were performed as described elsewhere. Digital image analysis Image acquisition, processing, and evaluation were performed as described elsewhere. Results Identification of two groups of B-CLL according to CD38 expression The purified malignant B-CLL cells from 52 patients were double stained with CD38 and CD19 monoclonal antibodies. Two groups of B-CLL, i.e. CD38 + and CD38 − B-CLL, were identified using the cut-off limit of 30%, already utilized in previous studies. CGH analysis Chromosome imbalances were detected by CGH. Two examples of merged CGH images and the relative mean profiles of ten metaphases with chromosomal imbalances are shown in Discussion Samples from 52 patients with typical B-CLL, diagnosed according to morphology and surface phenotype, were subjected to CGH analysis in order to obtain a comprehensive view of chromosomal gains and losses and to identify copy number aberrations specific for this pathology. To increase the sensitivity of CGH, we purified B-cells from the peripheral blood of B-CLL patients when there were less than 90% B cells. Twenty-one out of 52 (40%) patients showed chromosome imbalances; 11/21 had single imbalances, whereas the remaining 10 patients had two or more chromosome alterations. Thirty-three per cent of patients had received chemotherapy before cytogenetic analysis. The presence of patients subjected to therapy in the cohort can hardly be avoided in this kind of study. As an example, a recent study based on a large cohort of patients included a similar percentage of treated patients as did our study. Imbalances involving chromosomes 11, 12, 13 and 17 are among the most important factors in predicting survival: patients with 17p deletions are those with the worst prognosis, followed by patients with 11q deletion, those with 12q trisomy, and those with normal karyotypes, whereas patients with 13q deletions as the sole abnormality have the longest estimated survival times. This finding is possibly explained by the fact that our cohort included only patients with typical morphology/immunophenotype and that there were fewer patients with advanced stage disease. It has been demonstrated that atypical morphology and advanced stage disease correlate with loss in 17p. The B-CLL cases in this study could be subdivided into two groups according to the surface expression of CD38 by the malignant cells. This confirms previous findings from our laboratory and is also in line with data reported by others. The striking finding of this study was the clear correlation existing between the presence of chromosomal abnormalities and CD38 expression by the malignant cells. Thirteen out of 16 CD38 + patients also had chromosomal abnormalities, whereas, among the 36 CD38 − patients only 8 displayed chromosomal imbalances. These differences are highly significant (p=0.0001). Three out of the 8 CD38 − patients with chromosomal alterations had a loss in 13q as a single aberration, which generally correlates with a good prognosis, 3 patients had rare alterations, the prognostic value of which remains to be determined, while the remaining 2 patients had aberrations correlated with a poor prognosis (-11q; +12). Twelve of 13 CD38 + patients with chromosomal alterations displayed aberrations that are correlated with a poor clinical outcome (-11q; +12; -17p), whereas one patient had a gain in © F e r r a t a S t o r t i F o u n d a t i o n Chromosome aberrations and CD38 expression in B-CLL haematologica/journal of hematology vol. 88(07):july 2003 775 chromosome 3q, which is rarely found in B-CLL and is hence of undetermined prognostic value. Moreover, of the 10 patients with two or more chromosome imbalances (another marker of poor prognosis) detected in this study, 9 were within the CD38 + group. Remarkably, among the cases with the highest values of CD38 expression, 3 cases had simultaneous gain of chromosomes 12 and 18. Gain of chromosome 18 never appeared alone, but was always associated with gain of chromosome 12. Although this association has already been described in B-CLL by classical cytogenetic studies, 24,40 its significance and real frequency are not well documented. This is, in part, because most of the studies on chromosomal aberrations in large cohorts of B-CLL patients were performed with FISH using a panel of probes not including the chromosome 18 probe. Our data are in keeping with the recent observations that the unbalanced distribution of genomic aberrations in IgVH high mutation and low mutation subgroups might point toward a distinct biological background in such B-CLL subgroups and may in part, explain their different behaviors. 14 In the study by Kröber et al., 14 genomic aberrations and VH mutation status appeared to have a complementary role in estimating prognosis. Although CD38 expression has been proposed as an easily performed surrogate of VH mutational status analysis 4 its prognostic value is not completely clarified. Moreover, the relationship between CD38 expression and chromosomal aberrations has not been extensively studied. In a recent paper, Chevallier et al. Concerning the prognostic significance of CD38 expression in multivariate analysis, the authors suggested that a much larger group of patients was needed. Oscier et al. 36 showed that the mean survival of patients with loss in 17p was the shortest (47 months). In the present study the groups of patients with and without chromosomal alterations do not differ in terms of survival (not shown) probably since all but 2 patients are still alive. However, the simple patient in our cohort with loss in 17p at diagnosis was followed for only 1 year and was experiencing a poor clinical course. The different biological properties showed by B-CLL cells, including the expression of CD38, can help to explain the differences in the patients' outcomes. Recent studies, including those from our laboratory, 7,41 demonstrated that CD38 + B-CLL cells with unmutated VH/VL region genes have a viable IgM initiated signal transduction pathway. This pathway can lead to proliferation/differentiation or apoptosis depending on co-signals received by the cells in vitro. In contrast, most of the CD38 − mutated B-CLL cells do not respond to signals delivered to surface Ig. Therefore, the interaction between the cells and the environment via B-cell receptor is much less marked in CD38 − mutated cases than in CD38 + unmutated cases. These data suggest that CD38 + B-CLL cells are likely to be continuously stimulated via surface Ig. This is related to the fact that surface Ig, encoded by unmutated VH/VL genes, retain natural antibody activity and hence can react continuously with autoantigens in vivo. In the case of surface CD38-negative, mutated B-CLL cells, it is unlikely that the B-cell receptor can exert a promoting role on cell expansion since there is a not a viable IgM signal transducing pathway. Moreover, Ig encoded by mutated VH/VL genes rarely have natural antibody activity and, therefore, can rarely encounter the appropriate foreign antigen. Collectively, these considerations raise the issue of whether antigenic stimulation in B-CLL continues to exert a promoting effect on the growth of the malignant cells following transformation, and whether this is the reason for the clinical differences in B-CLL. Finally, it is unlikely that CD38 is solely a marker of cellular differentiation and clinical course. It is more probable that it also functions as a signaling molecule and, therefore may be directly involved in differences in disease severity. CD38 is known to play a role as an accessory molecule in B-cell receptor mediated signal transduction, 42,43 as well as regulating cell apoptosis in certain normal B-cell subsets. A number of conclusions can be drawn from this study. First, considering the increasing recognition of the importance of chromosome alterations in predicting the clinical outcome of B-CLL, the observation that chromosomal alterations are significantly more frequent within the CD38-positive cases lends further support to the prognostic value of the surface marker, CD38. Second, the finding that CD38 + cases can be subdivided into two groups (i.e. with and without chromosomal alterations) may lead to the delineation of further prognostic subsets of B-CLL. Third, the paucity of chromosomal alter- © F e r r a t a S t o r t i F o u n d a t i o

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicin

<it>In vitro </it>cell cultures obtained from different explants of <it>Corylus avellana </it>produce Taxol and taxanes

Abstract Background Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established. Results Calli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used. Conclusion Here we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a commercial source of Taxol and taxanes, both to be used as new therapeutic agents or as new precursors for Taxol semi-synthesis.</p

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%-40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP-ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity