The different banding patterns produced by restriction endonuclease digestion in mitotic chromosomes of the American and European eel

The detection of three classes of C-heterochromatin by in situ restriction endonuclease digestion allowed a karyotype differentiation between the American and the European eel.     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

Diverging trends of chronic bronchitis and smoking habits between 1998 and 2010

Background

No study has been carried out on the time trend in the prevalence of chronic bronchitis (CB) in recent years, despite its clinical and epidemiological relevance. We evaluated the trend in CB prevalence during the past decade among young Italian adults.

Methods

A screening questionnaire was mailed to general population samples of 20–44 year-old subjects in two cross-sectional surveys: the Italian Study on Asthma in Young Adults (ISAYA) (1998/2000; n = 18,873, 9 centres) and the screening stage of the Gene Environment Interactions in Respiratory Diseases (GEIRD) study (2007/2010; n = 10,494, 7 centres). CB was defined as having cough and phlegm on most days for a minimum of 3 months a year and for at least 2 successive years. The prevalence rates and the risk ratios (RRs) for the association between CB and each potential predictor were adjusted for gender, age, season of response, type of contact, cumulative response rate, and centre.

Results

CB prevalence was 12.5% (95% CI: 12.1-12.9%) in 1998/2000 and 12.6% (95% CI: 11.7-13.7%) in 2007/2010; it increased among never smokers (from 7.6 to 9.1%, p = 0.003), current light smokers (<15 pack-years; from 15.1 to 18.6%, p < 0.001), and unemployed/retired subjects (from 14.3 to 19.1%, p = 0.001). In this decade, the prevalence of current smoking decreased (from 33.6 to 26.9%, p < 0.001), whereas the prevalence of unemployment/premature retirement (from 5.3 to 6.0%, p = 0.005), asthma (from 5.0 to 6.2%, p = 0.003), and allergic rhinitis (from 19.5 to 24.5%, p < 0.001) increased. In both 1998/2000 and 2007/2010, the likelihood of having CB was significantly higher for women, current smokers, asthmatic patients, and subjects with allergic rhinitis. During this period, the strength of the association between CB and current heavy smoking (≥15 pack-years) decreased (RR: from 4.82 to 3.57, p = 0.018), whereas it increased for unemployment/premature retirement (from 1.11 to 1.53, p = 0.019); no change was observed for gender, asthma, and allergic rhinitis.

Conclusions

Despite the significant reduction in current smoking, CB prevalence did not vary among young Italian adults. The temporal pattern of CB prevalence can only be partly explained by the increase of unemployment/premature retirement, asthma and allergic rhinitis, and suggests that other factors could have played a role.     

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.     

Reference Charts for Fetal Cerebellar Vermis Height: A Prospective Cross-Sectional Study of 10605 Fetuses

Objective

To establish reference charts for fetal cerebellar vermis height in an unselected population.

Methods

A prospective cross-sectional study between September 2009 and December 2014 was carried out at ALTAMEDICA Fetal–Maternal Medical Centre, Rome, Italy. Of 25203 fetal biometric measurements, 12167 (48%) measurements of the cerebellar vermis were available. After excluding 1562 (12.8%) measurements, a total of 10605 (87.2%) fetuses were considered and analyzed once only. Parametric and nonparametric quantile regression models were used for the statistical analysis. In order to evaluate the robustness of the proposed reference charts regarding various distributional assumptions on the ultrasound measurements at hand, we compared the gestational age-specific reference curves we produced through the statistical methods used. Normal mean height based on parametric and nonparametric methods were defined for each week of gestation and the regression equation expressing the height of the cerebellar vermis as a function of gestational age was calculated. Finally the correlation between dimension/gestation was measured.

Results

The mean height of the cerebellar vermis was 12.7mm (SD, 1.6mm; 95% confidence interval, 12.7–12.8mm). The regression equation expressing the height of the CV as a function of the gestational age was: height (mm) = -4.85+0.78 x gestational age. The correlation between dimension/gestation was expressed by the coefficient r = 0.87.

Conclusion

This is the first prospective cross-sectional study on fetal cerebellar vermis biometry with such a large sample size reported in literature. It is a detailed statistical survey and contains new centile-based reference charts for fetal height of cerebellar vermis measurements.     

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.     

Multiple freshwater invasions of the tapertail anchovy (Clupeiformes: Engraulidae) of the Yangtze River

Freshwater fish evolved from anadromous ancestors can be found in almost all continents. The roles of paleogeographic events and nature selection in speciation process often are under focus of research. We studied genetic diversity of anadromous and resident tapertail anchovies (Coilia nasus species complex) in the Yangtze River Basin using 4,434 nuclear loci, and tested the history of freshwater invasion of C. nasus. We found that both C. brachygnathus and C. nasus were valid species, but the resident C. nasus taihuensis and the anadromous C. nasus were not different genetically based on Bayes factor species delimitation (BFD*). Maximum likelihood tree, Network, PCA and STRUCTURE analyses all corroborated the results of BFD*. Two independent freshwater invasion events of C. nasus were supported, with the first event occurring around 4.07 Ma and the second happened around 3.2 Ka. The time of the two freshwater invasions is consistent with different paleogeographic events. Estimation showed that gene flow was higher within ecotypes than between different ecotypes. F‐DIST analyses identified 120 disruptive outliers by comparing C. brachygnathus to anadromous C. nasus, and 21 disruptive outliers by comparing resident C. nasus to anadromous C. nasus. Nine outliers were found to be common between the two comparisons, indicating that independent freshwater invasion of C. nasus might involve similar molecular pathways. The results of this study suggest that adaptation to landlocked freshwater environment of migratory fish can evolve multiple times independently, and morphology of landlocked ecotypes may cause confusion in their taxonomy.     

Replication Banding in two Mediterranean Moray eels: Chromosomal Characterization and Comparison

Early and late replication bandings have been obtained by in vitro BrdU incorporation in the Mediterranean Muraenidae species Muraena helena and Gymnothorax unicolor, and used to characterize their karyotypes. A comparative analysis of the banding patterns allowed to point out high karyotype similarity as well as chromosome rearrangements that occurred in karyotype evolution between these species.