The achievement of boar sperm in vitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Voltage-dependent anion channel 2 is involved in in vitro capacitation of boar sperm.

Ion channels play an important role during sperm capacitation allowing the transport through plasma and mitochondrial membranes of specific molecules that are essential for the achievement of this physiologic status. Given that voltage-dependent anion channel 2 (VDAC2) is present in boar spermatozoa and is known to be involved in calcium transport in somatic cells, this study aimed at determining whether it is implicated in sperm capacitation and the acrosome reaction. With this purpose, boar spermatozoa were capacitated in vitro for 4 hr, and acrosome reaction was induced with progesterone for a further hour, with or without the presence of two VDAC2-inhibitors (erastin and olesoxime) at two different concentrations (10 and 100 µM). At different time points (0, 120, 240, 270 and 300 min), an aliquot was taken and sperm motility, membrane integrity and lipid disorder were evaluated using computer-assisted sperm analysis and flow cytometry. The addition of the two inhibitors resulted in opposite effects. While erastin 100 µM reduced the percentage of non-capacitated spermatozoa, the presence of olesoxime at the same concentration prevented the increase in membrane lipid disorder, which is a feature of sperm capacitation. Such prevention was concomitant with a maintaining effect on sperm membrane integrity evaluated through SYBR14/PI. Our results suggest that VDAC2 is involved in the regulation of sperm capacitation, despite the fact that the mechanisms through which erastin and olesoxime act are different.

Involvement of tumor suppressors PTEN and p53 in the formation of multiple subtypes of liposarcoma.

Liposarcoma (LPS) is a type of soft tissue sarcoma that mostly occurs in adults, and in humans is characterized by amplifications of MDM2 and CDK4. The molecular pathogenesis of this malignancy is still poorly understood and, therefore, we developed a mouse model with conditional inactivation of PTEN and p53 to investigate these pathways in the progression of the disease. We show that deletion of these two tumor suppressors cooperate in the formation of multiple subtypes of LPS (from well-differentiated LPS to pleomorphic LPS). In addition, progression of the tumors is further characterized by the expression of D cyclins and CDK4/6, which allow for continued cell division. Microarray analysis also revealed novel genes that are differentially expressed between different subtypes of LPS, which could aid in understanding the disease and to unravel potential new therapeutic targets.

TSPYL2 is an essential component of the REST/NRSF transcriptional complex for TGFß signaling activation.

REST/NRSF is a transcriptional repressor of neuronal genes that has been implicated in development and cancer. In epithelial tissues, REST acts as a tumor suppressor and in breast cancer, loss of REST is associated with disease recurrence and poor prognosis. Here, we identify TSPYL2 (also known as CDA1 and DENTT) as a novel component of the REST protein complex. We show that REST and TSPYL2 are regulators of TGFß signaling and that cell-cycle arrest induced by TGFß requires both REST and TSPYL2. Importantly, knockdown of REST or TSPYL2 resulted in transformation of human mammary epithelial cells. Mechanistically, we demonstrate that the TSPYL2/REST complex promotes TGFß signaling by repressing the expression of genes, such as the proto-oncogene neurotrophic tyrosine kinase receptor C (TrkC). These data provide insight into the role of REST as a tumor suppressor in epithelial tissues through the regulation of the TGFß pathway.

MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9.

BACKGROUND: The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease. METHODS: We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours. RESULTS: We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma. CONCLUSIONS: In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.

GAB2 induces tumor angiogenesis in NRAS-driven melanoma.

GAB2 is a scaffold protein with diverse upstream and downstream effectors. MAPK and PI3K signaling pathways are known effectors of GAB2. It is amplified and overexpressed in a variety of human tumors including melanoma. Here we show a previously undescribed role for GAB2 in NRAS-driven melanoma. Specifically, we found that GAB2 is co-expressed with mutant NRAS in melanoma cell lines and tumor samples and its expression correlated with metastatic potential. Co-expression of GAB2(WT) and NRAS(G12D) in melanocytes and in melanoma cells increased anchorage-independent growth by providing GAB2-expressing cells a survival advantage through upregulation of BCL-2 family of anti-apoptotic factors. Of note, collaboration of GAB2 with mutant NRAS enhanced tumorigenesis in vivo and led to an increased vessel density with strong CD34 and VEGFR2 activity. We found that GAB2 facilitiated an angiogenic switch by upregulating HIF-1α and VEGF levels. This angiogenic response was significantly suppressed with the MEK inhibitor PD325901. These data suggest that GAB2-mediated signaling cascades collaborate with NRAS-driven downstream activation for conferring an aggressive phenotype in melanoma. Second, we show that GAB2/NRAS signaling axis is non-linear and non-redundant in melanocytes and melanoma, and thus are acting independent of each other. Finally, we establish a link between GAB2 and angiogenesis in melanoma for the first time. In conclusion, our findings provide evidence that GAB2 is a novel regulator of tumor angiogenesis in NRAS-driven melanoma through regulation of HIF-1α and VEGF expressions mediated by RAS-RAF-MEK-ERK signaling.

Cryotolerance of in vitro-produced porcine blastocysts is improved when using glucose instead of pyruvate and lactate during the first 2 days of embryo culture.

The objective of the present study was to determine the effects of replacing glucose with pyruvate and lactate during the first 48 h of in vitro culture (IVC) in NCSU-23 medium on embryo development, embryo quality and survival of porcine blastocysts after vitrification. To this end, in vitro-produced (IVP) porcine oocytes were cultured with either glucose for 6 days (IVC-Glu) or pyruvate-lactate from Day 0 to Day 2 and then with glucose until Day 6 (IVC-PyrLac). Blastocysts were vitrified on Day 6 using the Cryotop device and, after warming, survival rate and the apoptosis index were evaluated after 24 h incubation in NCSU-23 medium. No significant differences were observed between IVC-Glu and IVC-PyrLac in terms of cleavage rate, blastocyst yield, total number of cells per blastocyst or the apoptosis index (1.82±0.75% vs 3.18±0.88%, respectively) of non-vitrified embryos. However, a significant increase was seen in hatching/hatched blastocysts in the IVC-PyrLac compared with IVC-Glu treatment group (12.71±1.20% vs 3.54±0.47%, respectively). Regardless of treatment, vitrification impaired the survival rate and the apoptosis index. When comparing both treatments after warming, the percentage of apoptotic cells was significantly higher for blastocysts in the IVC-PyrLac compared with IVC-Glu group (18.55±3.49% vs 9.12±2.17%, respectively). In conclusion, under the conditions of the present study, replacement of glucose with pyruvate-lactate during the first 48 h of culture resulted in a lower cryotolerance of IVP porcine embryos.

PAX7-FKHR fusion gene inhibits myogenic differentiation via NF-kappaB upregulation

OBJECTIVE: Alveolar rhabdomyosarcomas (ARMS) are characterised by a PAX3/7-FKHR translocation, which is presumed to promote a differentiation arrest in the myogenic lineage, in which setting secondary genetic events occur, resulting in sarcomagenesis. The aim of this study was to identify the mechanism by which PAX3/7-FKHR expression results in a myogenic differentiation block, as discrete from the secondary genetic events that complete the sarcomagenic process. METHODS: We performed a novel differential gene expression analysis comparing normal mesenchymal stem cells with previously generated non-tumorigenic mesenchymal stem cells expressing the PAX7-FKHR fusion gene, as well as with a known tumorigenic, PAX7-FKHR-expressing ARMS cell line, CW9019. RESULTS: This novel analysis uncovered the upregulation of the NF-kappaB pathway as a function of PAX3/7-FKHR expression, but distinct from the secondary sarcomagenic process; thus implicating NF-kappaB as a mediator of the PAX3/7-FKHR differentiation block. We further show that NF-kappaB activity is upregulated in PAX7-FKHR cells when compared to parental MSCs due to upregulation of the PI3K/AKT pathway. In addition we show that NF-kappaB inhibits myogenesis via activation of cyclinD1/ cdk4 complexes, which sequester MyoD1, a key myogenic transcription factor. CONCLUSIONS: Our results highlight the importance of the NF-kappaB pathway in myogenesis and sarcomagenesis and suggest that this pathway may be one of the potential therapeutic targets in the treatment of ARMS (AU)

Alternate PAX3 and PAX7 C-terminal isoforms in myogenic differentiation and sarcomagenesis

OBJECTIVE: Pax3 and Pax7 are closely related genes that are involved in commitment of cells to a myogenic lineage during skeletal muscle development and regeneration. Several Pax3 and Pax7 transcripts are expressed from the genes, generating different isoforms with potentially distinct DNA binding and transactivation properties. The aim of this study was to investigate the implication of Pax3 and Pax7 C-terminal isoforms during myogenic differentiation and tumorigenesis, since fusions involving these genes are commonly associated with alveolar rhabdomyosarcoma (ARMS). METHODS: Uncommitted (mouse mesenchymal stem cells, MSCs) and committed (C2C12) myogenic precursor cells were stably transfected with PAX3/FKHR and PAXC7/ FKHR fusion genes. We analysed gene and protein expression comparing the newly generated cells with the parental cells, to determine the functional importance of Pax3 and Pax7 C-terminal isoforms. RESULTS: We found that the transcript Pax3c was expressed at low levels in undifferentiated C2C12 and MSCs cells, but its expression levels increased considerably at later stages of differentiation. However, expression levels of Pax3d transcript increased only slightly after differentiation. Pax7 transcripts, present before differentiation in committed C2C12 cells, but absent in uncommitted MSCs, increased noticeably in MSCs after differentiation. We also found that the presence of PAX/FKHR fusions prevented both C2C12 and MSC cells from terminal myogenic differentiation and increased the expression of discrete endogenous Pax3/7 transcripts, in particular Pax3d and Pax7B. CONCLUSIONS: Our results suggest that both Pax3 and Pax7 transcripts are required for commitment of cells to the myogenic lineage, with each transcript having a distinct role. More specifically, the Pax3c isoform may be required for terminal myogenic differentiation whereas the Pax3d isoform may be involved in undifferentiated cell maintenance and/or proliferation (AU)

[Nursing professionals' reading habits; a study in a hospital in Granada]. / Hábitos de lectura en enfermería: estudio en un hospital de granada.

In all the hospital library standards, as well as in the existing Accreditation Norms for hospitals, a section is included which cites the distinct professionals who work at the institution which the library services must attend to provide for their scientific information needs. Among the sanitary collectives, nursing professionals are explicitly listed. Nonetheless, since the creation of hospital library services in Spain, many librarians have noticed that the nursing professionals, which compose the most numerous group in a hospital, have a very low library visitation rate in relationship to other collectives such as doctors in particular. Desiring to have objective data regarding library use, and not merely perceptions, the authors planned out a study in a large, 1.200 bed, hospital where more than 1.000 professionals comprise the nursing staff. The authors wanted to verify the nursing staff's needs for scientific information, their habits regarding their scientific information needs, and the possible difficulties which they encounter trying to acquire this type of information; furthermore, the authors wanted to know the nursing professionals' attitude towards this issue.