[Show abstract][Hide abstract] ABSTRACT: Interest in biochemistry of organoselenium compound has increased in the last decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of diphenyl diselenide (PhSe)2 (5 μmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Swiss male mice were divided into four experimental groups: control, (PhSe)2 (5 μmol/kg, subcutaneous administration), MeHg (40 mg/L, in tap water), and MeHg + (PhSe)2. After the treatment (21 days), the animals were killed and the cerebral cortex was analyzed. Electron microscopy indicated an enlarged and fused mitochondria leading to a reduced number of organelles, in the MeHg-exposed mice. Furthermore, cortical creatine kinase activity, a sensitive mitochondrial oxidative stress sensor, was almost abolished by MeHg. Subcutaneous (PhSe)2 co-treatment rescued from MeHg-induced mitochondrial alterations. (PhSe)2 also behaved as an enhancer of mitochondrial biogenesis, by increasing cortical mitochondria content in mouse-receiving (PhSe)2 alone. Mechanistically, (PhSe)2 (1 μM; 24 h) would trigger the cytoprotective Nrf-2 pathway for activating target genes, since astroglial cells exposed to the chalcogen showed increased content of hemeoxygenase type 1, a sensitive marker of the activation of this via. Thus, it is proposed that the (PhSe)2-neuroprotective effect might be linked to its mitoprotective activity.
Molecular and Cellular Biochemistry 03/2014; · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analysed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signalling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signalling pathways.
Experimental Cell Research 08/2013; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactotroph cells display morphological and functional heterogeneity, a feature which is closely related to the oestrogenic environment. In this study, we focused on sex-related differences linked to the proliferative and secretory responses of lactotrophs exposed to EGF in vitro. Furthermore, we addressed the involvement of the PKCε/ERK1/2 signalling pathway and the contribution of Pit-1 in the EGF actions in primary pituitary cultures from male and female rats. EGF promoted a differential proliferative activity on PRL cells, which was closely associated to the sex, as revealed by the uptake of bromodeoxyuridine (BrdU). In females, the mitogenic activity was up to nine times greater, whereas in males, the number of BrdU-labelled PRL cells was only doubled compared to control. However, in both models, EGF had a similar effectiveness in promoting PRL secretion. EGF also induced a significant increase in the PKCε, P -ERK 1/2, and Pit-1 protein levels, which were higher in females than in males. Pre-incubation with BIM blocked EGF-induced ERK 1/2 activation and Pit-1 expression. These results suggest a sexually dimorphic response of lactotroph cells to the proliferative effects of EGF, with the PKCε/ERK1/2 Pit-1 pathway being involved in this action.
[Show abstract][Hide abstract] ABSTRACT: The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.
Journal of molecular histology 02/2010; 40(5-6):417-25. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated.
The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy.
Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min.
We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.
Cellular Physiology and Biochemistry 01/2010; 26(2):135-46. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1-400x10(-8)M) and time periods (1-5h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100x10(-8)M) and GnRH (0.1 and 1x10(-9)M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4h of NEI incubation. NEI (100x10(-8)M) induced a significant LH secretion after 2h of stimulus, reaching a maximum response 4h later. A rapid and remarkable LH release was induced by NEI (400x10(-8)M) 1h after stimulus, attaining its highest level at 2h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10x10(-8)M), caused no action when used alone, its co-incubation with GnRH (1x10(-9)M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.
[Show abstract][Hide abstract] ABSTRACT: Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors.The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis.We also investigated the participation of p38, ERK1/2 and PKCδ in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKCδ in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats.In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKCδ, ERK1/2 and p38.
Toxicology and Applied Pharmacology 10/2009; · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.
Molecular and Cellular Endocrinology 08/2008; 289(1-2):77-84. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.
[Show abstract][Hide abstract] ABSTRACT: The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express
TRα1–β1 isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally
submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal
epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar
TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive
epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In
consonance to the immunocytochemical analysis, the expression of TRα1–β1 isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions.
Furthermore, TR expression of both α1 and β1 isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction
for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels
assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 microM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.
Cell and Tissue Research 02/2007; 327(1):121-32. · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clara cells are nonciliated secretory cells implicated in lung homeostasis by the synthesis of immunomodulatory and host defense products, being one of the most important the CC16 protein. In this study, we compared the effects of budesonide (BUD), an inhaled corticoid, on Clara cell biology and its ability to reverse morphofunctional changes induced in an allergic airway hyper-responsiveness mouse model. In normal mice, exposure to BUD induced morphological changes compatible with a state of maximal differentiation on CC16 positive cells which developed a prominent cupola filled up with numerous mitochondria rich in CYP2E1, a member of the cytochrome P450 family. Consequently, CYP2E1 expression raised significantly. Exposure to OVA provoked hypertrophy of Clara cells and an increment in their number per millimeter of basal membrane. These cells acquired a mucous cell phenotype characterized by a notorious expansion of the secretory granular content. Synthesis of CC16 was greatly up-regulated concurrent to the finding of MUC5AC expression and the increment of epidermal growth factor receptor (EGFR). Mitochondrial content decreased significantly with a consequent reduction in CYP2E1 expression. After BUD treatment of OVA-challenged animals, the majority of Clara cells regained their normal morphology and functional characteristics; CYP2E1 levels raised when compared to the OVA exposed group. The BUD potential to differentiate Clara cells appeared to be important for the regression of the profound changes generated by the allergic injury. These results demonstrated the wide range of stimuli that can modify different aspects of Clara cell biology, and highlighted the effects of budesonide as a modulator of P450 enzymes, which probably contributes to a complementary antiinflamatory activity.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.
Journal of Molecular Histology 11/2005; 36(8-9):447-54. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of IGF-1, 17 beta oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC alpha, epsilon and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two-threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 microM) or BIM (0.5 or 2 microM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 beta oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC alpha, epsilon and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.
[Show abstract][Hide abstract] ABSTRACT: The pituitary protein transcription factor (Pit-1) regulates the differentiation and proliferation of somatotrophs, lactotrophs, and thyrotrophs and the c-Myc oncoprotein plays a critical role in somatotroph and lactotroph differentiation. Both were involved in the genesis of pituitary tumors. The combined analysis of Pit-1 and c-Myc expression and the morphometric and biochemical parameters of the lactotroph population after treatment with estrogen for 7, 20, and 60 days provided new information on molecular mechanisms implicated in the formation of prolactinomas. Estrogen treatment for 7 days caused a significant proliferation of lactotrophs (70%) and this increase reached an additional 55% at 60 days. The proliferation of lactotrophs was concurrent with higher serum and pituitary prolactin levels. An augmentation of Pit-1 and c-Myc expression in both cytoplasmic and nuclear extracts after estrogen can be associated with lactotroph proliferation. Moreover, the multistep correlation analysis revealed that the expression of nuclear Pit-1 was the strongest predictor of prolactinoma development. Also the Pit-1 immunolocalization in nuclei of gonadotrophs suggests the activation of genes involved in transdifferentiation of gonadotroph to lactotroph. Therefore, the understanding of the Pit-1 function may help in the design of strategies to control the secretion and proliferation of pituitary tumors of the somatomammotrope lineage.
[Show abstract][Hide abstract] ABSTRACT: Triiodothyronine (T(3)) exerts most of its effects through nuclear thyroid hormone receptors (TRs) that bind mainly as heterodimers with retinoid-X receptors (RXRs) to thyroid hormone response elements in target genes. It is well known that T(3) activates the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis in rats. In turn, IGF-I inhibits the T(3)-induced GH production in cell cultures. The impact of IGF-I on T(3) action has only been partially explored. We have presented evidence that IGF-I feeds back to limit specific metabolic actions of T(3) in rat liver through a downregulation of nuclear TR number and its mRNA expression. We have also found that IGF-I injected to rats inhibited pituitary GH production. In this study we aimed at exploring whether the IGF-I-induced feedback loop on T(3)-action in the liver also operates in the pituitary gland. The mechanism of the liver TR mRNA reduction induced by IGF-I was also studied. We evaluated the effect of recombinant human (rh) IGF-I administration (240 microg/100 g of body weight subcutaneously every 12 hours for 48 hours) to adult male Wistar rats on TR and RXR proteins (Western blot) from pituitary, liver, brain, and thyroid and TR mRNA (Northern blot) from pituitary and liver. The transcriptional rate of liver TR gene (run-on assay) was also determined. In pituitary, TR protein and TR mRNA isoforms were reduced by rhIGF-I. No changes in TR proteins in brain and thyroid were observed. Nuclear run-on assay revealed that IGF-I reduced the TR gene transcriptional rate in liver. A significant increase in RXR proteins in liver and pituitary without changes in thyroid and brain was induced by IGF-I. In conclusion, these results indicate that in pituitary, IGF-I downregulates TR expression, similarly as previously found in liver. A reduced transcriptional rate of TR gene is implicated in the IGF-I effect on the liver. The increase in RXR protein levels may be also involved in the expression of T(3) specific actions in liver and pituitary.
[Show abstract][Hide abstract] ABSTRACT: In the present study the in vivo and in vitro effects of GHRP-5 on the PRL-releasing activity in correlation with the morphological changes of lactotroph cells and their transcriptional activity were evaluated. The in vivo treatment (12 micrograms/100 g BW/day for 3 days) of male rats with GHRP-5 does not induce any significant changes in serum PRL levels. In contrast, the addition of GHRP-5 to pituitary cell cultures increased significantly the release of PRL. This effect is enhanced in cell cultures of enriched lactotrophs, increasing significantly the secretion of PRL, the concentrations of which were 50% higher than that of untreated control cells. The administration of GHRP-5 provokes several changes in the fine structure of lactotrophs, compatible with an increased secretory activity. After the GHRP-5 treatment the different lactotroph subtypes persist but the subtype I displaying secretory granules of larger size (500-900 nm) and a significant development of the Golgi apparatus and RER were more frequently observed. These results can be correlated with a significant augmentation in PRL mRNA after the GHRP-5 treatment. In spite of that no variations in serum PRL levels were observed in vivo, following GHRP-5 treatment, the lactotroph population experienced evident fine structure modifications, concordant with an upsurge of PRL synthesis. These observations confirmed a direct action of GHRP-5 on receptors expressed by lactotrophs. The differential actions of GHRP-5 on in vivo and in vitro designs confirm a different effectiveness of this secretagogue to induce PRL secretion.
Biocell: official journal of the Sociedades Latinoamericanas de Microscopía Electronica ... et. al 05/2002; 26(1):49-60. · 0.58 Impact Factor