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Cancer cells are differentially dependent on the splicing machinery compared to normal untransformed cells. The splicing machinery thus represents a potential therapeutic target in cancer. To identify splicing factors important for prostate cancer cell (PCa) cell growth, we performed a parallel pooled shRNA screen on in vitro passaged cells and in vivo xenografted PCa tumor lines. Our screen revealed HNRNPM as a potential regulator of PCa cell growth. RNA- and eCLIP-sequencing data suggest that HNRNPM is bound to transcripts of key homeostatic genes and that loss of HNRNPM binding in a subset of these genes results in aberrant exon inclusion and exon back-splicing events in target transcripts. In both linear and circular mis-spliced transcripts, HNRNPM appears to preferentially bind to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching antisense oligonucleotides (SSOs) was sufficient to inhibit cell growth in HNRNPM expressing cells. This suggests that prostate cancer cell dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Taken together, our data reveal a role for HNRNPM in supporting prostate cancer cell fitness, and also as a potential therapeutic target in PCa.

The aim of “CISIA” project, funded by National Research Council, was to improve the valorization and sustainability of Southern Italy agrifood products. One of the area of application of this project is to genotype Mediterranean Italian Buffaloes breeding in order to detect the genes involved in fertility and reproductive seasonality. For this reason, our contribute to this project has been to perform physical maps using bovine artificial chromosomes (BAC) clones containing genes related to fecundity. BACs were selected taking in account the data available on the Bov Map database (http://locus. jouy.inra.fr/cgibin/bovmap/intro2.pl), considering their physical position and the data obtained from banding experiment s. Fluorescent in situ hybridization (FISH) was performed on three gene sequences: tumor necrosis factor-α (TNF), correlated to male fertility; signal transducer and activator of transcription 5A (STAT5A) important for its influence on milk production and reproduction activity; melatonin receptor 1A (MNTR1A) important for reproductive seasonality. BAC probes were hybridized on RB-banded of river buffalo (Bubalus bubalis, 2n=50, BBU), for the first time and to relate bovid species: cattle (Agerolese breed), sheep (Laticauda breed) and goat (Cilentana breed). TNF was assigned to BTA/CHI23q21-22, OAR20q21-22 and BBU 2p21-22; STAT5A was assigned to BTA/CHI19q17- 21, OAR11q17-21 and BBU3p15-21; MTNR1A was assigned to BTA/CHI27q14-15, OAR11q17-21 and BBU1p21-22, underling the high degree of chromosome homologies among Bovids and extending the cytogenetic maps of this economically important species.

Pig from Casertana and Siciliana breeds are two endangered breeds raised in the southern-Italy (Campania and Sicily regions, respectively) and characterized to have a black skin. Special projects are trying to save and characterize both of them by using several approaches. In this study we report the preliminary results we obtained after a cytogenetic investigation we performed by using both C- and R-banding techniques and the sister chromatid exchange (SCE) test to verify their chromosome stability under the environmental conditions. Fifty-two pigs from Casertana breed and 19 pigs from Siciliana breed were investigated. All animals from both breeds showed a normal karyotype, with the exception of two male pig from Siciliana breed which were found heterozygous carrier of rob(15;17) (2n=37, XY), probably being hybrids with the wild pig (2n=36) present in the Nebrodi mountains where this breed is raised in Sicily. SCE-test applied on 42 pigs from Casertana breed (22 males and 20 females) and 19 pigs from Siciliana breed (8 males and 11 females) revealed no statistical differences between the SCE-mean number in Casertana pig (7.13±3.20) than that (6.87±3.12) achieved in Siciliana pig. Statistical differences were found between males (7.26±3.38) and females (6.59±2.90) of Siciliana pig breed, as well as between females of Casertana (7.24±3.26) and Siciliana (6.59±2.90) breeds, while no statistical differences were found between males of the breeds, as well as between males and females of Casertana breed.

Phenylpropanoid glycosides (PPG), like other phenolic compounds, are powerful antioxidants. Beside phenolic compounds, verbascoside, shows the highest scavenger activity in the PPG and has high antioxidant power in comparison with other phenolic compounds. Previous studies by using in vitro exposure of human blood lymphocytes to verbascoside reported a significant increasing of chromosome fragility compared to control. In the present study four homogeneous groups of rabbits (six animals per group) were used to test in vivo the verbascoside by feeding the animals without Verbascoside and Licopene (control – group A), with lycopene (5 mg/Kg of feeding, group B), with verbascoside (5 mg/Kg of feeding, group C) with verbascoside and lycopene (5 mg/Kg of feeding each, group D). Peripheral blood cultures were performed in three different times: at 0, 40 and 80 days of the experiment. Two types of cell cultures were performed: without (normal cultures) for the AC-test (chromosome and chromatid breaks) and with BrdU (10 μg/ml), the latter added 26 h before harvesting, for the SCE-test. In the present study only data from SCE-test are presented. Mean number of SCEs were generally lower at both 40 and 80 days in groups B, C and D, compared with the same groups at zero day. In particular, they were statistically (P

To form three-dimensional capillary tubes, endothelial cells must establish contacts with the extracellular matrix that provides signals for their proliferation, migration, and differentiation. The transcription factor Fosl1 plays a key role in the vasculogenic and angiogenic processes as Fosl1 knockout embryos die with vascular defects in extraembryonic tissues. Here, we show that Fosl1(-/-) embryonic stem cells differentiate into endothelial cells but fail to correctly assemble into primitive capillaries and to form tube-like structures. FOSL1 silencing affects in vitro angiogenesis, increases cell adhesion, and decreases cell mobility of primary human endothelial cells (HUVEC). We further show that FOSL1 is a repressor of αv and β3 integrin expression and that the down-modulation of αvβ3 rescues the angiogenic phenotype in FOSL1-silenced HUVEC, while the ectopic expression of αvβ3 alone reproduces the phenotypic alterations induced by FOSL1 knockdown. FOSL1 represses the transcription of both αv and β3 integrin genes by binding together with JunD to their proximal promoter via the transcription factor SP1. These data suggest that FOSL1-dependent negative regulation of αvβ3 expression on endothelial cells is required for endothelial assembly into vessel structures.

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Cytogenetic maps are useful tools for several applications, such as the physical anchoring of linkage and RH maps or genome sequence contigs to specific chromosome regions or the analysis of chromosome rearrangements. Recently, a detailed RH map was reported in OAR1. In the present study, we selected 38 markers equally distributed in this RH map for identification of ovine genomic DNA clones within the ovine BAC library CHORI-243 using the virtual sheep genome browser and performed FISH mapping for both comparison of OAR1 and homoeologous chromosomes BBU1q-BBU6 and BTA1-BTA3 and considerably extending the cytogenetic maps of the involved species-specific chromosomes. Comparison of the resulting maps with human-identified homology with HSA2q, HSA3, HSA21 and HSA1q reveals complex chromosome rearrangements differentiating human and bovid chromosomes. In addition, we identified 2 new small human segments from HSA2q and HSA3q conserved in the telomeric regions of OAR1p and homoeologous chromosome regions of BTA3 and BBU6, and OAR1q, respectively. Evaluation of the present OAR1 cytogenetic map and the OAR1 RH map supports previous RH assignments with 2 main exceptions. The 2 loci BMS4011 and CL638002 occupy inverted positions in these 2 maps.

Donkey chromosomes were earlier characterized separately by C-, G- and R-banding techniques. However, direct comparisons between G- and R-banding patterns have still not been carried out in this species. The present study reports this comparison at the 450-band level by using replication G- and R-banding patterns. Two sets of synchronized lymphocyte cultures were set up to obtain early (GBA+CBA-banding) and late (RBA-banding) BrdU incorporation. Slides were stained with acridine orange and observed under a fluorescence microscope. Reverse GBA+CBA- and RBA-banded karyotypes at the 450-band level were constructed. To verify G- and R-banding patterns in some acrocentric chromosomes, sequential GBA+CBA/Ag-NORs and RBA/Ag-NORs were also performed. The results of CBA-banding patterns obtained in 12 animals from 2 breeds showed a pronounced polymorphism of heterochromatin, especially in EAS1q-prox. Ideogrammatic representations of G- and R-banded karyotypes were constructed using only one common G- and R-banding nomenclature. In the present study both G- and R-banding patterns and relative ideograms are presented as standard karyotype for this species at the 450-band level.

Polychlorodibenzodioxins (PCDDs) and related substances including polychlorodibenzofurans (PCDF) and the dioxin-like polychlorobiphenyls (DL-PCBs) are powerful mutagens that can enter the food chain as the result of a number of industrial processes, waste incineration or illegal waste burning. Cytogenetic tests could be useful to reveal the presence of such mutagens in the food chain by simply monitoring food producing species. In previous studies we studied sheep exposed to dioxins and found a pronounced chromosome fragility in exposed herds, compared to unexposed one (control) by using both SCE and chromosome abnormality (AC) test. In this study we applied the SCE-test in both cattle and river buffaloes reared in dioxin-contaminated areas, although the WHO-TEQ levels of bulk milk were much lower than those earlier detected in sheep milk. Peripheral blood cultures were performed in samples from 15 dairy cows showing average milk values over (18.56 pg/g of fat as WHO-TEQ) than those permitted (6.0 pg/g of dioxins+furans+PCBs as WHO-TEQ) and the results were compared with samples from 16 dairy cows (1.75 pg/g of fat as WHO-TEQ) used as control. Significantly (P < 0.01) higher mean number of SCE/cell (7.10 ± 2.8) were found in dioxin exposed cows compared to those measured in the controls (SCE/cell=5.24 ± 2.51). The same test was applied on 26 river buffaloes showing average values of WHO-TEQ over (21.79 pg/g of fat as WHO-TEQ) than those achieved in the milk mass of 26 river buffaloes (1.30 pg/g of fat as WHO-TEQ) used as control. Opposite to cattle, no significant differences were found between mean number of SCE/cell in exposed vs. non exposed river buffaloes (8.73 ± 3.32 vs. 8.79 ± 3.40).

International audience; Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISHmapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.

Presented herein is an updated sheep cytogenetic map that contains 452 loci (291 type I and 161 type II) assigned to specific chromosome bands or regions on standard R-banded ideograms. This map, which significantly extends our knowledge of the physical organization of the ovine genome, includes new assignments for 88 autosomal loci, including 74 type I loci (known genes) and 14 type II loci (SSRs/microsatellite marker/STSs), by FISH-mapping and R-banding. Comparison of the ovine map to the cattle and goat cytogenetic maps showed that common loci were located within homologous chromosomes and chromosome bands, confirming the high level of conservation of autosomes among ruminant species. Eleven loci that were FISH-mapped in sheep (B3GAT2, ASCC3, RARSL, BRD2, POLR1C, PPP2R5D, TNRC5, BAT2, BAT4, CDC5L and OLA-DRA) are unassigned in cattle and goat. Eleven other loci (D3S32, D1S86, BMS2621, SFXN5, D5S3, D5S68, CSKB1, D7S49, D9S15, D9S55 and D29S35) were assigned to specific ovine chromosome (OAR) bands but have only been assigned to chromosomes in cattle and goat.

International audience