Unusual Clinical Manifestations in a Mexican Patient with Sanfilippo B Syndrome.

We present an unusual Mexican patient affected with mucopolysaccharidosis type IIIB (MPS IIIB; also called Sanfilippo B syndrome, MIM #252920) bearing clinical features that have not previously been described for MPS IIIB (growth arrest, hypogonadotropic hypogonadism, and congenital heart disease). Chromosomal microarray analysis was useful in identifying runs of homozygosity at 17q11.1-q21.33 and supporting the diagnosis of an underlying autosomal recessive condition. Sanger sequencing of NAGLU (17q21.2, MIM*609701) allowed us to identify a pathogenic homozygous p.(Arg234Cys) genotype. This NAGLU allele could be related to that previously described in an Iberian MPS IIIB founder haplotype; results from the polymorphic marker D17S800 and rs2071046 led us to hypothesize that it may have been introduced to Mexico through the Spanish settlement. The analysis of a clinical exome sequencing ruled out other monogenic etiologies for the previously undescribed clinical MPS IIIB manifestations. Our findings contribute to further delineating the MPS IIIB phenotype and suggest possible phenotype-genotype correlations.

Salinomycin induces cell cycle arrest and apoptosis and modulates hepatic cytochrome P450 mRNA expression in HepG2/C3a cells.

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells' survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Mitochondrial Small Heat Shock Proteins Are Essential for Normal Growth of Arabidopsis thaliana.

Mitochondria play important roles in the plant stress responses and the detoxification of the reactive oxygen species generated in the electron transport chain. Expression of genes encoding stress-related proteins such as the mitochondrial small heat shock proteins (M-sHSP) is upregulated in response to different abiotic stresses. In Arabidopsis thaliana, three M-sHSPs paralogous genes were identified, although their function under physiological conditions remains elusive. The aim of this work is to uncover the in vivo function of all three M-sHSPs at the whole plant level. To accomplish this goal, we analyzed the phenotype, proteomic, and metabolic profiles of Arabidopsis knock-down lines of M-sHSPs (single, double, and triple knock-down lines) during normal plant growth. The triple knock-down plants showed the most prominent altered phenotype at vegetative and reproductive stages without any externally applied stress. They displayed chlorotic leaves, growth arrest, and low seed production. Concomitantly, they exhibited increased levels of sugars, proline, and citric, malic, and ascorbic acid, among other metabolites. In contrast, single and double knock-down plants displayed a few changes in their phenotype. A redundant function among the three M-sHSPs is indicated by the impairment in vegetative and reproductive growth associated with the simultaneous loss of all three M-sHSPs genes. The triple knock-down lines showed alteration of proteins mainly involved in photosynthesis and antioxidant defense compared to the control plants. On the other hand, heat stress triggered a distinct cytosolic response pattern and the upregulation of other sHSP members, in the knock-down plants. Overall, depletion of all three M-sHSPs in Arabidopsis severely impacted fundamental metabolic processes, leading to alterations in the correct plant growth and development. These findings expand our knowledge about the contribution of organelle-specific M-sHSPs to healthy plant growth under non-stress conditions.

Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells.

INTRODUCTION: Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and through intra and extrapancreatic nerves, even during the early stages of the disease, a phenomenon called perineural invasion (PNI). Thus, we sought to determine the effects of the simultaneous expression of soluble forms of GAS1 and PTEN (tGAS1 and PTEN-L) inhibiting tumor growth and invasiveness. MATERIALS AND METHODS: We employed a lentiviral system to simultaneously express tGAS1 and PTEN-L; in order to determine the effects of the treatments, cell viability and apoptosis as well as the expression of the transgenes by ELISA and intracellular signaling as ascertained by the activation of AKT and ERK1/2 were measured; cell invasiveness was determined using a Boyden chamber assay; and the effects of the treatment were measured in vivo in a mouse model. RESULTS: In the present work, we show that the combined treatment with tGAS1 and PTEN-L inhibits the growth of pancreatic cancer cells, by reducing the activities of both AKT and ERK 1/2, decreases cell invasiveness, and restrains tumor growth in a mouse model. CONCLUSION: The combined administration of tGAS1 and PTEN-L could be a valuable adjunct therapy for the treatment of pancreatic cancer.

Significance of pro-angiogenic estrogen metabolites in normal follicular development and follicular growth arrest in polycystic ovary syndrome.

STUDY QUESTION: Do alterations in pro- and anti-angiogenic estrogen metabolites in follicular fluid (FF) contribute to the follicular growth arrest and anovulation associated with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: FF of PCOS women with anovulation have reduced levels of pro-angiogenic estrogen metabolites (EMs) and vascular endothelial growth factor (VEGF) compared to that of fertile women with regular menstrual cycles, but exogenous gonadotropins increase the pro-angiogenic EMs and VEGF levels in PCOS women. WHAT IS KNOWN ALREADY: PCOS is characterized by the arrest of follicular development that leads to chronic anovulation. Follicular arrest is generally associated with elevated plasma levels of luteinizing hormone (LH), androgens and anti-Mullerian hormone (AMH). There is also reduced angiogenesis in the follicles of PCOS women compared to those of normal cycling women. It is known that angiogenesis is a critical factor during follicular development. We and other investigators have explored the role of EMs in ovarian angiogenesis, particularly in human corpus luteum function, showing that 4-hydroxyestrone (4-OHE1) and 16-ketoestradiol (16-kE2) have pro-angiogenic effects while 2-methoxyestradiol (2-ME2) and 2-methoxyestrone (2-ME1) have anti-angiogenic effects. Additionally, 2-hydroxyestradiol (2-OHE2), which is produced in the ovary, has proliferative and pro-angiogenic properties. We hypothesized that EMs could be involved in angiogenesis necessary for ovarian follicular development in fertile women, and that dysregulation of these factors may contribute to follicular arrest in PCOS. The relationship between EMs, VEGF and AMH in the pathophysiology of follicular arrest in PCOS has not been previously studied at a follicular level in anovulatory women without ovulation induction. STUDY DESIGN, SIZE, DURATION: This is a comparative experimental study of serum and FF collected from different sized follicles (antral ˂10 mm and dominant ˃16 mm) of women with and without ovarian stimulation. The study included women with regular menstrual cycles who were proven to be fertile (n = 20) and PCOS women with follicular arrest who were candidates for ovarian drilling (n = 17), as well as other patients requiring ovarian stimulation, i.e. control women undergoing IVF for male factor infertility (n = 12) and PCOS women undergoing IVF (n = 17). In vitro studies were carried out on granulosa-lutein cells (GCs) obtained from subsets of women undergoing IVF for male factor infertility (n = 6) and PCOS women undergoing IVF (n = 6). GCs were maintained in culture for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Intrafollicular estradiol, estrone and EMs concentrations were determined by high performance liquid chromatography-mass spectrometry. Testosterone in serum was measured by RIA, and LH, FSH and sex hormone-binding globulin in serum were measured with IRMA kits. AMH was determined in serum and FF by enzyme linked immunosorbant assay (ELISA). VEGF levels were measured in FF and conditioned medium by ELISA. Conditioned medium were obtained from cultured GCs. The angiogenic potential was assessed by in vitro angiogenic assays. MAIN RESULTS AND THE ROLE OF CHANCE: Pro-angiogenic EMs (4-OHE1, 16-kE2 and 2-OHE2) and VEGF were lower in FF of antral follicles of PCOS women with follicular arrest compared those of fertile women with ovulatory cycles (P < 0.05). In contrast, higher concentrations of AMH were found in FF of antral follicles from PCOS women with follicular arrest compared to those of fertile women with ovulatory cycles (P < 0.05). Exogenous gonadotropins used in IVF increased pro-angiogenic EMs and VEGF production in PCOS women, reaching similar profiles compared to control women receiving gonadotropins in their IVF treatment for male factor infertility. The pro-angiogenic EM 2-OHE2 increased the angiogenic potential and VEGF levels of GCs from PCOS women compared to the basal condition (P < 0.05). These findings suggest that there is a role for pro-angiogenic EMs in the control of follicular VEGF production. LIMITATIONS, REASONS FOR CAUTION: The limitations include the possibility that in vitro analysis of GCs might not reflect the in vivo mechanisms involved in the pro-angiogenic action of 2-OHE2 since GCs obtained at the time of oocyte retrieval belong to a very early stage of the luteal phase and might not be representative of GCs during follicular growth. Therefore, our findings do not conclusively rule out the possibility that other in vivo mechanisms also account for defective angiogenesis observed in PCOS. WIDER IMPLICATIONS OF THE FINDINGS: The present study highlights the significance of EMs, angiogenic factors and AMH and their interaction in the pathophysiology of follicular development in PCOS. This study provides new insights into the role of pro-angiogenic factors in follicular arrest in PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Vascular calcification and growth arrest specific protein 6 levels in chronic renal failure / Calcificación vascular y detención de crecimiento de proteína específica 6 niveles en insuficiencia renal crónica

INTRODUCTION: Cardiovascular disease is the main cause of mortality and morbidity in chronic renal failure. It's known that vascular calcification (VC) and carotid intima media thickness (CIMT) are strongly associated with cardiovascular diseases. Growth arrest specific protein 6 (Gas6) is a vitamin K-dependent protein and regulates various processes such as proliferation, cell survival, migration and inflammation. Gas6 is known to protect endothelial cells and vascular smooth muscle cells against apoptosis by inhibiting Bcl-2 induced Caspase 3 activation. The relationship between Gas6 and cardiovascular diseases has been demonstrated in many mouse models and cell cultures. However, there are conflicting reports whether Gas6 levels are increasing or decreasing in human studies of diabetic and/or chronic renal failure. In present study the aim was to examine plasma Gas6 levels and its relation with CIMT and coronary artery calcification score (CACS) in chronic kidney disease (CKD) patients. METHODS: Total of 137 patients of which 32 chronic hemodialysis and 105 predialysis patients as well as 73 healthy controls were enrolled in the study. Human Gas6 levels in serum samples were studied by ELISA method. CIMT was measured by ultrasonography. CACS was measured by multislice computed tomography. RESULTS: The mean age was 54.37±16.61 years in dialysis group, 55.20±14.80 years in predialysis group and 53.26±9.04 years in control group. Serum creatinine was 0.78±0.16 mg/dl in the control group and 1.96±1.64 mg/dl in the predialysis group and 5.94±1.55 mg/dl in the dialysis group. 24 hours urine protein levels were significally higher in the dialysis group than the predialysis and the control group. CIMT values were similar in predialysis and dialysis groups. These values were significantly higher than the control group. Although CACS was higher in dialysis group than predialysis and control group, the results were not statistically significant since the distribution range was very wide. Gas6 was 98.84±53.32 ng/mL in the control group and statistically higher than the dialysis (63.85±38.92 ng/mL) and the predialysis groups (54.96±38.49 ng/mL) (p=0.001). Gas6 levels were lower in diabetic patients than non-diabetics (53.69±35.26 ng/mL, 69.26±47.50 ng/mL, p=0.023, respectively). Negative correlation was detected between Gas6 and age, BMI, CACS, carotid IMT and proteinuria. In the logistic regression analysis, Gas6 remained significantly associated with BMI, CIMT and proteinuria. CONCLUSION: In our study, a negative correlation of Gas6 with BMI, CACS, CIMT and proteinuria and lower Gas6 levels in diabetic patients support that decreased Gas6 levels in chronic renal failure may have a role in vascular calcification through altered glucose tolerance, chronic inflammation, endothelial dysfunction and increased apoptosis. Our study has an importance because it is the first study showing a relation between Gas6 and proteinuria, CACS and carotid IMT in patients with chronic renal failure

INTRODUCCIÓN: La enfermedad cardiovascular es la principal causa de mortalidad y morbilidad en la insuficiencia renal crónica. Se sabe que la calcificación vascular (CV) y el grosor de la íntima-media de la carótida (CIMT, por sus siglas en inglés) están vinculados de forma muy estrecha con enfermedades cardiovasculares. La proteína específica del gen 6 de la detención de crecimiento (Gas6) es una proteína dependiente de la vitamina K y regula diversos procesos, como la proliferación, la supervivencia celular, la migración y la inflamación. La proteína Gas6 es conocida por proteger las células endoteliales y las células musculares lisas vasculares contra la apoptosis mediante la inhibición de la activación de la caspasa-3 inducida por la proteína Bcl-2. Se ha demostrado la relación entre la Gas6 y las enfermedades cardiovasculares en muchos modelos de ratones y cultivos celulares. Sin embargo, existen informes contradictorios acerca de si los niveles de Gas6 aumentan o disminuyen en estudios de humanos con insuficiencia renal crónica y/o diabética. En este estudio, el objetivo fue examinar los niveles plasmáticos de Gas6 y su relación con el CIMT y la puntuación de calcificación de las arterias coronarias (CACS, por sus siglas en inglés) en pacientes con enfermedad renal crónica (ERC). MATERIAL Y MÉTODOS: Un total de 137 pacientes fueron incluidos en el estudio, de los cuales 32 estaban en hemodiálisis crónica, 105 en prediálisis, y 73 pacientes representaban controles sanos. Se esudiaron los niveles de Gas6 en muestras de suero mediante el método ELISA. El CIMT se midió por medio de ecografía. La CACS se midió mediante tomografía computarizada multicorte. RESULTADOS: La edad media fue de 54,37 ± 16,61 años en el grupo de diálisis; 55,20 ± 14,80 años en el grupo de prediálisis, y 53,26 ± 9,04 años en el grupo de control. La creatinina sérica fue de 0,78 ± 0,16 mg/dl en el grupo de control; 1,96 ± 1,64 mg/dl en el de prediálisis, y 5,94 ± 1,55 mg/dl en el de diálisis. Las concentraciones de proteína en orina de 24 horas fueron significativamente más altas en el grupo de diálisis que en los de prediálisis y control. Los valores del CIMT fueron similares en los grupos de prediálisis y de diálisis. Estos valores fueron considerablemnete más altos que en el grupo de control. Aunque la CACS fue más alta en el grupo de diálisis que en los otros dos, los resultados no fueron estadísticamente significativos, ya que el rango de distribución fue muy amplio. La proteína Gas6 fue de 98,84 ± 53,32 ng/ml en el grupo de control y estadísticamente más alta que en los grupos de diálisis (63,85 ± 38,92 ng/ml) y de prediálisis (54,96 ± 38,49 ng/ml) (p = 0,001). Los niveles de Gas6 fueron más bajos en los pacientes diabéticos que en los no diabéticos (53,69 ± 35,26 ng/ml; 69,26 ± 47,50 ng/ml, [p = 0,023], respectivamente). Se detectó una correlación negativa entre la proteína Gas6 y la edad, el IMC, la CACS, el CIMT y la proteinuria. En el análisis de regresión logística, la Gas6 se mantuvo estrechamente relacionada con el IMC, el CIMT y la proteinuria. CONCLUSIÓN: En nuestro estudio, la correlación negativa de Gas6 con IMC, CACS, CIMT y proteinuria, y los niveles más bajos de Gas6 en pacientes diabéticos sustentan la idea de que la disminución de los niveles de Gas6 en la insuficiencia renal crónica puede jugar un papel en la calcificación vascular a través de la tolerancia alterada a la glucosa, la inflamación crónica, la disfunción endotelial y el aumento de la apoptosis. La importancia de nuestro estudio radica en que es el primero que muestra una relación entre la Gas6 y la proteinuria, la CACS y el CIMT en pacientes con insuficiencia renal crónica

Expression of Gas1 in Mouse Brain: Release and Role in Neuronal Differentiation.

Growth arrest-specific 1 (Gas1) is a pleiotropic protein that induces apoptosis of tumor cells and has important roles during development. Recently, the presence of two forms of Gas1 was reported: one attached to the cell membrane by a GPI anchor; and a soluble extracellular form shed by cells. Previously, we showed that Gas1 is expressed in different areas of the adult mouse CNS. Here, we report the levels of Gas1 mRNA protein in different regions and analyzed its expressions in glutamatergic, GABAergic, and dopaminergic neurons. We found that Gas1 is expressed in GABAergic and glutamatergic neurons in the Purkinje-molecular layer of the cerebellum, hippocampus, thalamus, and fastigial nucleus, as well as in dopaminergic neurons of the substantia nigra. In all cases, Gas1 was found in the cell bodies, but not in the neuropil. The Purkinje and the molecular layers show the highest levels of Gas1, whereas the granule cell layer has low levels. Moreover, we detected the expression and release of Gas1 from primary cultures of Purkinje cells and from hippocampal neurons as well as from neuronal cell lines, but not from cerebellar granular cells. In addition, using SH-SY5Y cells differentiated with retinoic acid as a neuronal model, we found that extracellular Gas1 promotes neurite outgrowth, increases the levels of tyrosine hydroxylase, and stimulates the inhibition of GSK3ß. These findings demonstrate that Gas1 is expressed and released by neurons and promotes differentiation, suggesting an important role for Gas1 in cellular signaling in the CNS.

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model.

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF-RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.

Pao Pereira Extract Suppresses Castration-Resistant Prostate Cancer Cell Growth, Survival, and Invasion Through Inhibition of NFκB Signaling.

Pao extract, derived from bark of Amazonian tree Pao Pereira, is commonly used in South American medicine. A recent study showed that Pao extract repressed androgen-dependent LNCaP prostate cancer cell growth. We hypothesize that Pao extract asserts its anticancer effects on metastatic castration-resistant prostate cancer (CRPC) cells. Pao extract suppressed CRPC PC3 cell growth in a dose- and time-dependent manner, through induction of apoptosis and cell cycle arrest. Pao extract treatment induced cell cycle inhibitors, p21 and p27, and repressed PCNA, Cyclin A and Cyclin D1. Furthermore, Pao extract also induced the upregulation of pro-apoptotic Bax, reduction of anti-apoptotic Bcl-2, Bcl-xL, and XIAP expression, which were associated with the cleavage of PARP protein. Moreover, Pao extract treatment blocked PC3 cell migration and invasion. Mechanistically, Pao extract suppressed phosphorylation levels of AKT and NFκB/p65, NFκB DNA binding activity, and luciferase reporter activity. Pao inhibited TNFα-induced relocation of NFκB/p65 to the nucleus, NFκB/p65 transcription activity, and MMP9 activity as shown by zymography. Consistently, NFκB/p65 downstream targets involved in proliferation (Cyclin D1), survival (Bcl-2, Bcl-xL, and XIAP), and metastasis (VEGFa, MMP9, and GROα/CXCL1) were also downregulated by Pao extract. Finally, forced expression of NFκB/p65 reversed the growth inhibitory effect of Pao extract. Overall, Pao extract induced cell growth arrest, apoptosis, partially through inhibiting NFκB activation in prostate cancer cells. These data suggest that Pao extract may be beneficial for protection against CRPC.