Label‐free and non‐invasive discrimination of HaCaT and melanoma cells in a co‐culture model by hyperspectral confocal reflectance microscopy

A novel hyperspectral confocal microscopy method to separate different cell populations in a co‐culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non‐invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

Set of hyperspectral images of melanoma‐keratinocytes co‐culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label‐free spectral identification of cell populations (right).     

Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder,glioblastoma and pancreatic cancer cell lines

Carbonic anhydrase IX/XII (CA IX/XII), are cell-surface enzymes typically expressed by cancer cells as a form of adaptation to hypoxia and acidosis. It has been widely reported that these proteins play pivotal roles in cancer progression fostering cell migration, aggressiveness and resistance to first line chemo- and radiotherapies. CA IX has emerged as a promising target in cancer therapy and several approaches and families of compounds were characterised in the attempt to find optimal targeting by inhibiting of the high catalytic activity of the enzyme. In the present work, different cell lines representing glioblastoma, bladder and pancreatic cancer have been exploited to compare the inhibitory and antiproliferative effect of primary sulphonamide acetazolamide (AAZ), the Phase Ib/II clinical grade sulphonamide SLC-0111, and a membrane-impermeant positively charged, pyridinium-derivative (C18). New hints regarding the possibility to exploit CA inhibitors in these cancer types are proposed.     

Influence of physical preconditioning on the responsiveness of rat pulmonary artery after pulmonary ischemia/reperfusion

The aim of this work was to evaluate the effect of physical preconditioning in the responsiveness of rat pulmonary rings submitted to lung ischemia/reperfusion (IR). Wistar rats were divided into three groups: Sedentary sham-operated (SD/SHAM); sedentary submitted to ischemia/reperfusion (SD/IR) and trained submitted to ischemia/reperfusion (TR/IR) animals. Exercise training consisted in sessions of 60 min/day running sessions, 5 days/week for 8 weeks. Left pulmonary IR was performed by occluding for 90 min and reperfusing for 120 min. After that, pulmonary arteries were isolated and concentration-response curves to acetylcholine (ACh), histamine (HIST), sodium nitroprusside (SNP), phenylephrine and U46619 were obtained. Neither potency (-log EC(50)) nor maximal responses (E(max)) were modified for ACh and HIST in all groups. On the other hand, the potency for SNP was significantly increased in TR/IR group (8.23+/-0.06) compared to SD/IR group (7.85+/-0.04). Contractile responses mediated by a-adrenergic receptor were markedly decreased in IR groups (SD/IR: 6.75+/-0.06 and TR/IR: 6.62+/-0.04) compared to SD/SHAM (7.33+/-0.05). No changes were seen for the U46619 in all groups. In conclusion, the present study shows that exercise training has no protective actions in the local blood vessel where the IR process takes place.     

Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data.

BACKGROUND: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification. METHODS: By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA. RESULTS: CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95- whereas CD127-, CD95+ phenotypes were found at higher frequencies in people with advanced age. CONCLUSIONS: Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects.     

Characterization of virulence factors and phylogenetic group determination of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from diarrheic and non-diarrheic calves from Brazil

This study aimed to detect virulence factors, pathovars, and phylogenetic groups of Escherichia coli strains obtained from feces of calves with and without diarrhea up to 70 days old and to determine the association between occurrence of diarrhea, phylogenetic groups, and pathovars. Phylo-typing analysis of the 336 E. coli strains isolated from calves with Clermont method showed that 21 (6.25 %) belong to phylogroup A, 228 (67.85 %) to phylogroup B1, 2 (0.6 %) to phylogroup B2, 5 (1.49 %) to phylogroup C, 57 (16.96 %) to phylogroup E, and 3 (0.9 %) to phylogroup F. Phylogroup D was not identified and 20 strains (5.95 %) were assigned as “unknown.” The distribution of phylogenetic groups among pathovars showed that NTEC belong to phylogroups B1 (17) and C (4); EPEC to phylogroups B1 (6) and E (8); STEC to phylogroups A (5), B1 (56), B2 (2), C (1), and E (15); EHEC to phylogroups B1 (95) and E (5); and ETEC to phylogroups A (3), B1 (7), and E (10). The EAST-1 strains were phylogroups A (13), B1 (47), E (19), and F (3); E. coli strains of “unknown” phylogroups belonged to pathovars EPEC (1), EHEC (2), STEC (7), and EAST-1 strains (6). ETEC was associated with diarrhea (P = 0.002). Our study did not find association between the phylogenetic background and occurrence of diarrhea (P = 0.164) but did find some relationship in phylogenetic group and pathovar. The study showed that EHEC and STEC are classified as phylogroup B1, EAST-1 phylogroup A, ETEC, and EPEC phylogroup E.     

Genetic polymorphisms of Fas (CD95) and FasL (CD178) in human longevity: studies on centenarians

Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism, at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.