PI3K inhibition by BKM120 results in anti-proliferative effects on corticotroph tumor cells.

PURPOSE: Cushing's disease is associated with significant morbidity; thus, additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway, which plays regulatory role in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, on cell viability, apoptosis, cell cycle phase distribution, and ACTH production in mouse corticotroph tumor cells. METHODS: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using an MTS-based assay. Apoptosis was evaluated by Annexin V staining. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Ccnd1, Ccne1, Cdk2, Cdk4, Myc, and Rb1) was assessed by qPCR. Protein expression of p27, total and phosphorylated Akt was assessed by Western blot. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. RESULTS: Treatment with BKM120 decreased AtT-20/D16v-F2 cell viability, induced a G0/G1 cell cycle arrest, reduced the phosphorylation of Akt at Serine 473, and increased p27 expression. Furthermore, BKM120 treatment diminished ACTH levels in the cell culture supernatants. CONCLUSION: In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in anti-proliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing's disease.

Spiritual well-being and quality of life are impaired in chronic urticaria.

SUMMARY: Background. Patients with chronic urticaria (CU) often report an impaired quality of life (QoL). Although a positive effect of addressing spirituality in health care has been proved in several chronic diseases, its potential role in CU has received no attention. Objective. We aim to evaluate spirituality and QoL in CU subjects. Methods. In a single-centre observational study, 100 CU subjects were investigated using Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being (FACIT-Sp-12) scale, Chronic Urticaria Quality of life Questionnaire (CU-Q2oL) and Urticaria Control Test (UCT). Results. Of 100 subjects, 82 were female and 18 were male. It was observed that subjects with poorly controlled CU presented FACIT Sp-12 meaning/peace (p = 0.004) significantly lower, and CU-Q2oL (p less than 0.0001) significantly higher (worst QoL) than subjects with controlled CU. There was no difference in the FACIT Sp-12 faith (p = 0.43) between groups. There was moderate direct correlation between FACIT Sp-12 faith and FACIT Sp-12 meaning/peace (r = 0.483; p less than 0.0001; n = 100). There was a significant strong inverse correlation between the CU-Q2oL and the UCT (r = - 0.762; p less than 0.0001; n = 100). No correlation was found between the FACIT Sp-12 faith and CU-Q2oL, neither with UCT. Conclusions. No study has ever investigated the role of spirituality in managing patients with urticaria. Our findings support the impact of poorly controlled urticaria in spiritual well-being and QoL. Therefore, clinicians should pay more attention to spirituality among CU patients. We suggest that urticaria guidelines should include specific recommendations on spirituality assessment.

The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L.

Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis.

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas--FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

A novel polar-based human face recognition computational model

Motivated by a recently proposed biologically inspired face recognition approach, we investigated the relation between human behavior and a computational model based on Fourier-Bessel (FB) spatial patterns. We measured human recognition performance of FB filtered face images using an 8-alternative forced-choice method. Test stimuli were generated by converting the images from the spatial to the FB domain, filtering the resulting coefficients with a band-pass filter, and finally taking the inverse FB transformation of the filtered coefficients. The performance of the computational models was tested using a simulation of the psychophysical experiment. In the FB model, face images were first filtered by simulated V1- type neurons and later analyzed globally for their content of FB components. In general, there was a higher human contrast sensitivity to radially than to angularly filtered images, but both functions peaked at the 11.3-16 frequency interval. The FB-based model presented similar behavior with regard to peak position and relative sensitivity, but had a wider frequency band width and a narrower response range. The response pattern of two alternative models, based on local FB analysis and on raw luminance, strongly diverged from the human behavior patterns. These results suggest that human performance can be constrained by the type of information conveyed by polar patterns, and consequently that humans might use FB-like spatial patterns in face processing.

Parathyroid hormone secretion in chronic human endogenous hypercortisolism

Osteoporosis is a common manifestation of Cushing's syndrome, but the mechanisms responsible for this abnormality have not been defined. With the objective of analyzing parathyroid hormone (PTH) secretion in chronic hypercortisolism (CH), we evaluated 11 healthy subjects and 8 patients with CH, 6 with Cushing's disease and 2 with adrenal adenoma. These volunteers were submitted to tests of PTH stimulation through hypocalcemia (EDTA), PTH suppression through hypercalcemia (iv and oral calcium), and evaluation of bone mineral density (BMD) by DEXA. During the test of PTH stimulation, the calcium and magnesium concentrations of the normal and CH groups were similar. Patients with CH showed an increased PTH response to the hypocalcemic stimulus compared to controls. PTH values were significantly higher in the CH group at 70 (17.5 ± 3.5 vs 10.2 ± 1.3 pmol/l, P = 0.04), and 120 min (26.1 ± 5.9 vs 11.3 ± 1.9 pmol/l, P = 0.008) of EDTA infusion. The area under the curve for PTH during EDTA infusion was also significantly higher in patients with CH than in normal subjects (1867 ± 453 and 805 ± 148 pmol l-1 2 h-1, P = 0.02). During the test of PTH suppression, calcium, magnesium and PTH levels of the patients with hypercortisolism and controls were similar. BMD was decreased in patients with hypercortisolism in the spine (0.977 ± 0.052 vs 1.205 ± 0.038 g/cm² in controls, P<0.01). In conclusion, our results show that subjects with CH present decreased bone mass mainly in trabecular bone. The use of dynamic tests permitted the detection of increased PTH secretion in response to a hypocalcemic stimulus in CH patients that may probably be involved in the occurrence of osteoporosis in this state

[Study of the frequency of diabetes mellitus and glucose intolerance in patients with cystic fibrosis] / Estudo da freqüência de diabetes mellitus e intolerância à glicose em pacientes com fibrose cística.

OBJECTIVE: To evaluate the frequency of diabetes mellitus and glucose intolerance in patients with cystic fibrosis treated at the Pediatric Gastroenterology Service of HC-FMRP-USP. METHODS: A cross-sectional analytical study was conducted on a group of 25 patients with mucoviscidosis who were followed up at HC-FMRP-USP. Oral glucose tolerance tests (OGTT) were performed, with simultaneous determination of glycemia and insulinemia. Areas under the curve were obtained for glycemia (G) and insulinemia (I) and the I/G ratio was calculated and correlated with the duration of clinical manifestation and pancreatic exocrine function. RESULTS: Five patients presented alterations: one was diabetic and four had glucose intolerance and/or hyperinsulinemia. There was a direct correlation between the area under the curve for insulinemia and the duration of mucoviscidosis. A significant inverse correlation was also observed between the area under the curve for insulinemia and I/G ratio, and number of enzyme capsules/kg/day. CONCLUSIONS: The frequency of alterations in glucose homeostasis observed in patients with mucoviscidosis was higher than in the population at large (20% of the total sample and 33% of the group of patients with glycemia and insulinemia on OGTT). Therefore, it is important that glucose tolerance tests be performed systematically in patients with mucoviscidosis so that metabolic abnormalities can be early detected, and proper treatment can be initiated.

Neural cell classification by wavelets and multiscale curvature.

A new approach to automatic classification of retinal ganglion cells using multiscale techniques including the continuous wavelet transform, curvature, and standard pattern recognition techniques is described. Each neural cell is represented by its outer contour, and the wavelet transform is calculated from the complex signal defined by the aforementioned contour, leading to the so-called W-representation (Antoine et al. 1996). The normalized multiscale wavelet energy (NMWE) is used to define a set of shape measures associated with the number of details of the shape for a broad range of spatial scales. Next, the more discriminating NMWE coefficients are chosen through a feature ordering technique and fed to statistical classifiers. In addition, the normalized multiscale bending energy (NMBE) is discussed as a means of neural shape description for classification purposes based on the multiscale curvature, i.e. the curvegram, of the neural contour. It is shown that both shape descriptors are suitable for shape classification, presenting similar classification performance. In fact, NMBE has a slightly better recognition rate than NMWE in our experiments. On the other hand, NMWE is less computationally expensive than NMBE, presenting also the potentially useful property of allowing the use of more suitable different analyzing wavelets, depending on the problem under consideration. Therefore, both measures are related and provide a good framework for the design of neural cell description and classification. The methods described in this work have been successfully applied to the classification of two classes of cat retinal ganglion cells, namely alpha and beta (henceforth referred as alpha-cells and beta-cells, respectively), and three statistical classifiers were considered: minimum-distance, k-nearest neighbours and maximum likelihood. The mean recognition rates are near 90%, which is superior to the other shape measures considered. It is argued here that the proposed technique can be adopted as a new general methodology for multiscale shape analysis and recognition, being applicable also to other problems in biological shape characterization in neuroscience and general biomedical image analysis. In the context of analysis of shape complexity, the multiscale energies are coherent with subjective judgements by humans.