A novel nuclear function for the centrosomal serine/threonine kinase Nek2

Numerose chinasi che regolano il ciclo centrosomale sono frequentemente regolate in maniera aberrante nelle cellule neoplastiche. Cambiamenti nella loro espressione possono portare ad alterazioni nella duplicazione del centrosoma quindi aneuploide. I tumori delle cellule germinali testicolari sono caratterizzati da amplificazione dei centrosomi la cui causa è ancora largamente sconosciuta. Abbiamo individuato una nuova chinasi centrosomale, Nek2, che risulta up-regolata nei seminomi testicolari in maniera specifica e ne abbiamo caratterizzato la sua funzione nelle cellule germinali neoplastiche. Una scoperta inaspettata del nostro studio è stata la presenza di Nek2 nel nucleo delle cellule germinali di seminoma testicolare, localizzazione ritrovata anche nelle cellule di linea derivanti dai seminomi: le Tcam-2. Inoltre tale localizzazione è stata ritrovata nelle cellule germinali da cui i seminomi derivano: le cellule germinali primordiali e gli spermatogoni più indifferenziati. Tali risultati suggeriscono un ruolo di Nek2 come marcatore di staminalità delle cellule germinali maschili, mantenuto anche nella trasformazione neoplastica. Abbiamo inoltre dimostrato che la localizzazione nucleare di Nek2 non è una caratteristica peculiare dei seminomi testicolari ma è principalmente localizzata nel nucleo in diversi tipi di tumore. Nelle cellule tumorali inoltre Nek2 è presente in splicing speckles dove associa con numerosi fattori di splicing come la proteina SR (ASF/SF2), hnRNP (A1, F and H), e la proteina STAR Sam68. Il nostro studio dimostra che Nek2 è in grado di fosforilare Sam68 e di modularne l’attività di splicing. Questi risultati identificano un nuovo ruolo nucleare per Nek2Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication and aneuploidy. In addition, many centrosomal protein kinases participate to other aspects of cell cycle progression. Testicular germ cell tumors (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. We have discovered that the centrosomal kinase Nek2 is overexpressed in testicular seminomas and we have characterized its function in neoplastic germ cells. One unexpected finding of our study was the nuclear localization of Nek2 in germ cells of patients. The same nuclear localization was observed in the seminoma cell line Tcam-2. We found that Nek2 was localized in the nucleus also in undifferentiated embryonal male primordial germ cells (PGCs) and in spermatogonial stem cells from post-natal testis. These results suggest that nuclear Nek2 is a novel marker of the undifferentiated stage of male germ cells that is maintained in testicular seminomas, but not in other TGCTs. The nuclear localization of Nek2 is not a unique feature of testicular seminomas, we show that Nek2 is mainly distributed in the nucleus of cancer cells from other tissues, including breast, prostate and colon cancer cells. The subnuclear distribution of Nek2 in speckles closely resembled that of many regulators of pre-mRNA splicing. We found that Nek2 physically associates with several splicing factors, such as SR proteins (ASF/SF2), hnRNPs (A1, F and H), and the STAR protein Sam68. We focused our study on Sam68 because this splicing regulator is also up-regulated in breast and prostate carcinomas like Nek2. Our study shows that Sam68 is also overexpressed in testicular seminomas but not in other TGCTs, like Nek2. Moreover, Nek2 phosphorylates Sam68 and affects Sam68-dependent splicing of CD44v5 pre-mRNA, an alternatively spliced form of the receptor, frequently altered in cancer cells, that promotes cell proliferation and invasiveness. These results identify a novel nuclear function of Nek2 and suggest that modulation of alternative splicing events by this kinase can contribute to neoplastic transformatio

A novel nuclear function for the centrosomal serine/threonine kinase Nek2

Numerose chinasi che regolano il ciclo centrosomale sono frequentemente regolate in maniera aberrante nelle cellule neoplastiche. Cambiamenti nella loro espressione possono portare ad alterazioni nella duplicazione del centrosoma quindi aneuploide. I tumori delle cellule germinali testicolari sono caratterizzati da amplificazione dei centrosomi la cui causa è ancora largamente sconosciuta. Abbiamo individuato una nuova chinasi centrosomale, Nek2, che risulta up-regolata nei seminomi testicolari in maniera specifica e ne abbiamo caratterizzato la sua funzione nelle cellule germinali neoplastiche. Una scoperta inaspettata del nostro studio è stata la presenza di Nek2 nel nucleo delle cellule germinali di seminoma testicolare, localizzazione ritrovata anche nelle cellule di linea derivanti dai seminomi: le Tcam-2. Inoltre tale localizzazione è stata ritrovata nelle cellule germinali da cui i seminomi derivano: le cellule germinali primordiali e gli spermatogoni più indifferenziati. Tali risultati suggeriscono un ruolo di Nek2 come marcatore di staminalità delle cellule germinali maschili, mantenuto anche nella trasformazione neoplastica. Abbiamo inoltre dimostrato che la localizzazione nucleare di Nek2 non è una caratteristica peculiare dei seminomi testicolari ma è principalmente localizzata nel nucleo in diversi tipi di tumore. Nelle cellule tumorali inoltre Nek2 è presente in splicing speckles dove associa con numerosi fattori di splicing come la proteina SR (ASF/SF2), hnRNP (A1, F and H), e la proteina STAR Sam68. Il nostro studio dimostra che Nek2 è in grado di fosforilare Sam68 e di modularne l’attività di splicing. Questi risultati identificano un nuovo ruolo nucleare per Nek2Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication and aneuploidy. In addition, many centrosomal protein kinases participate to other aspects of cell cycle progression. Testicular germ cell tumors (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. We have discovered that the centrosomal kinase Nek2 is overexpressed in testicular seminomas and we have characterized its function in neoplastic germ cells. One unexpected finding of our study was the nuclear localization of Nek2 in germ cells of patients. The same nuclear localization was observed in the seminoma cell line Tcam-2. We found that Nek2 was localized in the nucleus also in undifferentiated embryonal male primordial germ cells (PGCs) and in spermatogonial stem cells from post-natal testis. These results suggest that nuclear Nek2 is a novel marker of the undifferentiated stage of male germ cells that is maintained in testicular seminomas, but not in other TGCTs. The nuclear localization of Nek2 is not a unique feature of testicular seminomas, we show that Nek2 is mainly distributed in the nucleus of cancer cells from other tissues, including breast, prostate and colon cancer cells. The subnuclear distribution of Nek2 in speckles closely resembled that of many regulators of pre-mRNA splicing. We found that Nek2 physically associates with several splicing factors, such as SR proteins (ASF/SF2), hnRNPs (A1, F and H), and the STAR protein Sam68. We focused our study on Sam68 because this splicing regulator is also up-regulated in breast and prostate carcinomas like Nek2. Our study shows that Sam68 is also overexpressed in testicular seminomas but not in other TGCTs, like Nek2. Moreover, Nek2 phosphorylates Sam68 and affects Sam68-dependent splicing of CD44v5 pre-mRNA, an alternatively spliced form of the receptor, frequently altered in cancer cells, that promotes cell proliferation and invasiveness. These results identify a novel nuclear function of Nek2 and suggest that modulation of alternative splicing events by this kinase can contribute to neoplastic transformatio

PDE5 inhibitors in type 2 diabetes cardiovascular complications

Pharmacological inhibition of Phosphodiesterase type 5 (PDE5) proved its efficacy treating several pathological conditions, such as erectile dysfunction and pulmonary hypertension. Nowadays, its benefits on cardiovascular diseases are well documented, particularly in the treatment of type 2 diabetes (T2DM)-related cardiovascular complications. In this context, treatment of T2DM with PDE5 inhibitors, such as sildenafil, tadalafil or vardenafil ameliorates endothelial dysfunction both in patients and animal models through an augmented flow mediated dilation rate and an up-regulation of endothelial markers; it also reduces the inflammatory state by down-regulating inflammatory cytokines expression and improves diabetic cardiomyopathy and ischemia-reperfusion injury mainly through the activation of NO-cGMP-PKG pathway. The present review summarizes the state of art on PDE5 inhibition in the treatment of cardiovascular complications in T2DM

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

Carbon balance and energy fluxes of a Mediterranean crop

This paper is based on the analysis of a long-term mass (carbon dioxide, water vapour) and energy (solar radiation) balance monitoring programme carried out during years 2010 and 2012 in an irrigated orange orchard in Sicily, using the Eddy Covariance (EC) method. Orange (Citrus sinensis L.) is one of the main fruit crops worldwide and its evergreen orchard may have a great potential for carbon sequestration, but few data are currently available. In the study, the role of the orchard system in sequestering atmospheric CO2 was analyzed, thus contributing to assess the carbon balance of the specie in the specific environment.Vertical energy fluxes of net radiation, soil heat, sensible heat and latent heat fluxes were measured at orchard scale by EC. Evapotranspiration (ET) values were compared with upscaled transpiration data determined by the sap flow heat pulse technique, evidencing the degree of correspondence between instantaneous transpirational flux at tree level and the micrometeorological measurement of ET at orchard level

Age-related curves of AMH using the Gen II, the picoAMH and the Elecsys assays in women with polycystic ovary syndrome

CONTEXT: Several challenges still exist to adopt the anti-Müllerian hormone (AMH) as a marker of polycystic ovary morphology (PCOM), as included in the recently updated international guideline. Although different evaluations of age- and assay-specific reference ranges have been published in the last years, these studies have mainly been conducted in normo-ovulatory or infertile women.OBJECTIVE: To develop an age-specific percentile distribution of AMH in patients with polycystic ovary syndrome (PCOS) measured by three different assays.DESIGN: Retrospective cross-sectional study.PATIENTS: 2,725 women aged 20 to 40 years with PCOS diagnosis were included.INTERVENTION (S): Serum AMH measurement by the Gen II (Beckman Coulter), the picoAMH (Ansh Labs), and the Elecsys (Roche) assays.MAIN OUTCOME MEAUSRE (S): Age-specific centile curves for all the assays and correlations between AMH, clinical, hormonal, and ultrasound characteristics.RESULTS: Age-related nomograms for the 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles of AMH were calculated using the LMS method for all the assays. AMH levels were significantly different between PCOS phenotypes. AMH levels were positive correlated to luteinizing hormone (LH), LH/follicular stimulating hormone (FSH) ratio, testosterone, androstenedione, free androgen index, mean follicular number, and mean ovarian volume.CONCLUSIONS: To our knowledge this is the first study reporting age specific percentile nomograms of serum AMH levels measured by the Gen II, the picoAMH and the Elecsys assays in a large population of PCOS women. These findings may help to interpret AMH levels in PCOS patients and facilitate the use of AMH as a diagnostic tool across age ranges.</p

Diagnostic value of qualitative and strain ratio elastography in the differential diagnosis of non-palpable testicular lesions

The purpose of this study was to evaluate prospectively the accuracy of qualitative and strain ratio elastography (SE) in the differential diagnosis of non-palpable testicular lesions. The local review board approved the protocol and all patients gave their consent. One hundred and six patients with non-palpable testicular lesions were consecutively enrolled. Baseline ultrasonography (US) and SE were correlated with clinical and histological features and ROC curves developed for diagnostic accuracy. The non-palpable lesions were all ≤1.5 cm; 37/106 (34.9%) were malignant, 38 (35.9%) were benign, and 31 (29.2%) were non-neoplastic. Independent risk factors for malignancy were as follows: size (OR 17.788; p = 0.002), microlithiasis (OR 17.673, p &lt; 0.001), intralesional vascularization (OR 9.207, p = 0.006), and hypoechogenicity (OR, 11.509, p = 0.036). Baseline US had 89.2% sensitivity (95% CI 74.6-97.0) and 85.5% specificity (95% CI 75.0-92.8) in identifying malignancies, and 94.6% sensitivity (95% CI 86.9-98.5) and 87.1% specificity (95% CI 70.2-96.4) in discriminating neoplasms from non-neoplastic lesions. An elasticity score (ES) of 3 out of 3 (ES3, maximum hardness) was recorded in 30/37 (81.1%) malignant lesions (p &lt; 0.001). An intermediate score of 2 (ES2) was recorded in 19/38 (36.8%) benign neoplastic lesions and in 22/31 (71%) non-neoplastic lesions (p = 0.005 and p = 0.001 vs. malignancies). None of the non-neoplastic lesions scored ES3. Logistic regression analysis revealed a significant association between ES3 and malignancy (χ2 = 42.212, p &lt; 0.001). ES1 and ES2 were predictors of benignity (p &lt; 0.01). Overall, SE was 81.8% sensitive (95% CI 64.8-92.0) and 79.1% specific (95% CI 68.3-88.4) in identifying malignancies, and 58.6% sensitive (95% CI 46.7-69.9) and 100% specific (95% CI 88.8-100) in discriminating non-neoplastic lesions. Strain ratio measurement did not improve the accuracy of qualitative elastography. Strain ratio measurement offers no improvement over elastographic qualitative assessment of testicular lesions; testicular SE may support conventional US in identifying non-neoplastic lesions when findings are controversial, but its added value in clinical practice remains to be proven

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment

Does a Very Short Length of Abstinence Improve Assisted Reproductive Technique Outcomes in Infertile Patients with Severe Oligo-Asthenozoospermia?

In recent years, a growing number of studies seem to support the beneficial effects of a very short abstinence period on sperm parameters, especially in patients with oligo-asthenozoospermia (OA). On this basis, the aim of this study was to evaluate the effects of a short period of abstinence (1 h) on intracytoplasmic sperm injection (ICSI) outcomes in infertile patients with severe OA. We performed a retrospective study on 313 ICSI cycles in which couples were divided into two different groups based on sperm parameters of the male partners. Group 1 included normozoospermic men or male partners with a mild OA (n = 223). Group 2 included male partners with severe OA (n = 90). They were asked to provide a second consecutive ejaculation after 1 h from the first one. The best ejaculate was used to perform ICSI. We found a significant increase of total (p < 0.001) and progressive motility (p < 0.001) in the second ejaculate of patients of Group 2 compared with those of the first one. Spermatozoa of the second ejaculate were chosen for ICSI for all patients in Group 2. We found statistically significant improvement of clinical pregnancy rate (p = 0.001) and embryo quality (p = 0.003) in couples in Group 2 compared to those of Group 1. No statistically significant difference was found in fertilization, implantation, live birth delivery, and miscarriage rates between the two groups. Therefore, a second semen sample collected after a very short time-interval in patients with severe OA allowed us to obtain significantly higher clinical pregnancy rate with improved embryo quality compared to normozoospermic men or patients with mild OA. Fertilization, implantation, live birth delivery, and miscarriage rates were similar between the two groups. The present study shows that a second consecutive ejaculate could represent a simple strategy to obtain better sperm parameters and assisted reproductive technology (ART) outcomes in infertile patients with mild-severe OA