Behavioral and genetic color vision evaluation of an albino male capuchin monkey (Sapajus apella).

Albinism is a rare phenotype that affects the pigmentation in eyes, hair, and skin. The effects of albinism in color vision are still unclear. Our study aimed to evaluate the color vision phenotype and genotype of an albino capuchin monkey. An adult albino male capuchin monkey (Sapajus apella) had the L and M opsin gene analyzed, and was trained in a behavioral task of color discrimination. Color discrimination thresholds were determined along 20 chromatic axes around the background chromaticity. A color discrimination ellipse was drawn by interpolation among these thresholds. The albino monkey's behavioral color discrimination ellipse showed poor discrimination along the red-green axis indicating a deutan phenotype. Genetic analysis revealed only the presence of the L gene in the albino monkey. This result did not differ from that obtained with ten previously tested non-albino monkeys. Behavioral and molecular analyses agreed that the albino capuchin monkey had color vision similar to that of non-albino dichromat monkeys, suggesting no influence of albinism on color discrimination.

Daily activity patterns influence retinal morphology, signatures of selection, and spectral tuning of opsin genes in colubrid snakes.

BACKGROUND: Morphological divergences of snake retinal structure point to complex evolutionary processes and adaptations. The Colubridae family has a remarkable variety of retinal structure that can range from all-cone and all-rod to duplex (cone/rod) retinas. To explore whether nocturnal versus diurnal activity is responsible for constraints on molecular evolution and plays a role in visual opsin spectral tuning of colubrids, we carried out molecular evolution analyses of the visual opsin genes LWS, RH1, and SWS1 from 17 species and performed morphological analyses. RESULTS: Phylogenetic reconstructions of the RH1 and LWS recovered major clades characterized by primarily diurnal or primarily nocturnal activity patterns, in contrast with the topology for SWS1, which is very similar to the species tree. We found stronger signals of purifying selection along diurnal and nocturnal lineages for RH1 and SWS1, respectively. A blue-shift of the RH1 spectral peak is associated with diurnal habits. Spectral tuning of cone opsins did not differ among diurnal and nocturnal species. Retinas of nocturnal colubrids had many rows of photoreceptor nuclei, with large numbers of rods, labeled by wheat germ agglutinin (WGA), and two types of cones: large cones sensitive to long/medium wavelengths (L/M) and small cones sensitive to ultra-violet/violet wavelengths (UV/VS). In contrast, retinas of diurnal species had only one row of photoreceptor nuclei, with four types of cones: large and double L/M cones, small UV/VS cones, and a second group of small cones, labeled by WGA. CONCLUSIONS: For LWS gene, selection tests did not confirm different constraints related to activity pattern. For SWS1, stronger purifying selection in nocturnal lineages indicates divergent evolutionary pressures related to the activity pattern, and the importance of the short wavelength sensitivity at low light condition. Activity pattern has a clear influence on the signatures of selection and spectral tuning of RH1, with stronger purifying selection in diurnal lineages, which indicates selective pressure to preserve rhodopsin structure and function in pure-cone retinas. We suggest that the presence of four cone types in primarily diurnal colubrids might be related to the gain of color discrimination capacity.

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition.

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients' samples and by in silico analysis on an independent patient cohort from Taylor's platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients' risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.

A microRNA code for prostate cancer metastasis.

Although the development of bone metastasis is a major detrimental event in prostate cancer, the molecular mechanisms responsible for bone homing and destruction remain largely unknown. Here we show that loss of miR-15 and miR-16 in cooperation with increased miR-21 expression promote prostate cancer spreading and bone lesions. This combination of microRNA endows bone-metastatic potential to prostate cancer cells. Concomitant loss of miR-15/miR-16 and gain of miR-21 aberrantly activate TGF-ß and Hedgehog signaling, that mediate local invasion, distant bone marrow colonization and osteolysis by prostate cancer cells. These findings establish a new molecular circuitry for prostate cancer metastasis that was validated in patients' cohorts. Our data indicate a network of biomarkers and druggable pathways to improve patient treatment.

BTG2 loss and miR-21 upregulation contribute to prostate cell transformation by inducing luminal markers expression and epithelial-mesenchymal transition.

Prostate cancer is one of the leading causes of cancer-related death in men. Despite significant advances in prostate cancer diagnosis and management, the molecular events involved in the transformation of normal prostate cells into cancer cells have not been fully understood. It is generally accepted that prostate cancer derives from the basal compartment while expressing luminal markers. We investigated whether downregulation of the basal protein B-cell translocation gene 2 (BTG2) is implicated in prostate cancer transformation and progression. Here we show that BTG2 loss can shift normal prostate basal cells towards luminal markers expression, a phenotype also accompanied by the appearance of epithelial-mesenchymal transition (EMT) traits. We also show that the overexpression of microRNA (miR)-21 suppresses BTG2 levels and promotes the acquisition of luminal markers and EMT in prostate cells. Furthermore, by using an innovative lentiviral vector able to compete with endogenous mRNA through the overexpression of the 3'-untranslated region of BTG2, we demonstrate that in prostate tumor cells, the levels of luminal and EMT markers can be reduced by derepression of BTG2 from microRNA-mediated control. Finally, we show that the loss of BTG2 expression confers to non-tumorigenic prostate cells ability to grow in an orthotopic murine model, thus demonstrating the central role of BTG2 downregulaton in prostate cancer biology.

Systemic in vivo lentiviral delivery of miR-15a/16 reduces malignancy in the NZB de novo mouse model of chronic lymphocytic leukemia.

Similar to human chronic lymphocytic leukemia (CLL), the de novo New Zealand Black (NZB) mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and in sera when compared with control lentivirus treatment. More importantly, mice treated with the miR-expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects, and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.

Control of tumor and microenvironment cross-talk by miR-15a and miR-16 in prostate cancer.

The interaction between cancer cells and microenvironment has a critical role in tumor development and progression. Although microRNAs regulate all the major biological mechanisms, their influence on tumor microenvironment is largely unexplored. Here, we investigate the role of microRNAs in the tumor-supportive capacity of stromal cells. We demonstrated that miR-15 and miR-16 are downregulated in fibroblasts surrounding the prostate tumors of the majority of 23 patients analyzed. Such downregulation of miR-15 and miR-16 in cancer-associated fibroblasts (CAFs) promoted tumor growth and progression through the reduced post-transcriptional repression of Fgf-2 and its receptor Fgfr1, which act on both stromal and tumor cells to enhance cancer cell survival, proliferation and migration. Moreover, reconstitution of miR-15 and miR-16 impaired considerably the tumor-supportive capability of stromal cells in vitro and in vivo. Our data suggest a molecular circuitry in which miR-15 and miR-16 and their correlated targets cooperate to promote tumor expansion and invasiveness through the concurrent activity on stromal and cancer cells, thus providing further support to the development of therapies aimed at reconstituting miR-15 and miR-16 in advanced prostate cancer.

Green fluorescent protein incorporation by mouse myoblasts may yield false evidence of myogenic differentiation of human haematopoietic stem cells.

AIMS: Haematopoietic CD34+ stem cells are able to differentiate into skeletal muscle, a potentially invaluable tool for treating degenerative diseases such as muscular dystrophy. However, some studies argue that the differentiative potential of these cells might have been overestimated. In vitro studies provide a controlled environment in which to investigate this point. METHODS: CD34+ stem cells from human peripheral blood, labelled with green fluorescent protein (GFP), were co-cultured with mouse myogenic C2C12 cells. The functional properties of mononucleated GFP+ cells were determined using electrophysiological techniques and were related to protein profiling determined by immunofluorescence staining and single-cell RT-PCR. Mouse mesoangioblasts co-cultured with human myotubes provided methodological controls. RESULTS: After 2-4 days, mononucleated adherent GFP+ cells showed acetylcholine-evoked current responses, typical of myogenic cells, as if stem cells had integrated into the host environment. In contrast to this hypothesis, human nuclei could not be detected in adherent GFP+ cells by immunofluorescence. Moreover, single-cell RT-PCR showed that adherent GFP+ cells responsive to acetylcholine expressed mouse markers while loose unresponsive GFP+ cells were of human origin. The transcripts of the human alpha1 subunit of the acetylcholine muscle receptor were not amplified in co-cultures. CONCLUSION: Single-cell analysis of functional properties combined with other markers revealed that, under the co-culture conditions used, GFP was transferred from human CD34+ stem cells to C2C12 myoblasts by mechanisms unrelated to myogenic stem cell differentiation. Our results emphasize the need for careful controls using several markers when investigating the myogenic differentiation of circulating stem cells.

A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.

Lentiviral transduction of human postnatal skeletal (stromal, mesenchymal) stem cells: in vivo transplantation and gene silencing.

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus.

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 microg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 microg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 +/- 393 cells/mm2) compared to control (1886 +/- 892 cells/mm2; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 +/- 56 cells/mm2 (2 microg/g) and 845 +/- 82 cells/mm2 (6 microg/g), also lower than control (1312 +/- 31 cells/mm2; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 microg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo.

Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the 'advanced' generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the 'advanced' generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy in vitro against the third-generation of the same vector. Here we report that 'advanced' generation lentiviral vectors infected most (>80%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three- to four-fold lower. Moreover, 'advanced' generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus, 'advanced' generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte.

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response.

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.

Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1.

The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.