Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a ""flipflop'' phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Hyperbaric Oxygen Prevents Early Death Caused by Experimental Cerebral Malaria

Background: Cerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis. Methodology/Principal Findings: C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O(2), 3.0 ATA, 1-2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-alpha, IFN-gamma and IL-10 mRNA levels and percentage of gamma delta and alpha beta CD4(+) and CD8(+) T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB)dysfunction and hypothermia. Conclusions/Significance: The data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.

Gene Expression Complex Networks: Synthesis, Identification, and Analysis

Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdos-Renyi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabasi-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference method was sensitive to average degree k variation, decreasing its network recovery rate with the increase of k. The signal size was important for the inference method to get better accuracy in the network identification rate, presenting very good results with small expression profiles. However, the adopted inference method was not sensible to recognize distinct structures of interaction among genes, presenting a similar behavior when applied to different network topologies. In summary, the proposed framework, though simple, was adequate for the validation of the inferred networks by identifying some properties of the evaluated method, which can be extended to other inference methods.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Inference of gene regulatory networks from time series by Tsallis entropy

Background: The inference of gene regulatory networks (GRNs) from large-scale expression profiles is one of the most challenging problems of Systems Biology nowadays. Many techniques and models have been proposed for this task. However, it is not generally possible to recover the original topology with great accuracy, mainly due to the short time series data in face of the high complexity of the networks and the intrinsic noise of the expression measurements. In order to improve the accuracy of GRNs inference methods based on entropy (mutual information), a new criterion function is here proposed. Results: In this paper we introduce the use of generalized entropy proposed by Tsallis, for the inference of GRNs from time series expression profiles. The inference process is based on a feature selection approach and the conditional entropy is applied as criterion function. In order to assess the proposed methodology, the algorithm is applied to recover the network topology from temporal expressions generated by an artificial gene network (AGN) model as well as from the DREAM challenge. The adopted AGN is based on theoretical models of complex networks and its gene transference function is obtained from random drawing on the set of possible Boolean functions, thus creating its dynamics. On the other hand, DREAM time series data presents variation of network size and its topologies are based on real networks. The dynamics are generated by continuous differential equations with noise and perturbation. By adopting both data sources, it is possible to estimate the average quality of the inference with respect to different network topologies, transfer functions and network sizes. Conclusions: A remarkable improvement of accuracy was observed in the experimental results by reducing the number of false connections in the inferred topology by the non-Shannon entropy. The obtained best free parameter of the Tsallis entropy was on average in the range 2.5 <= q <= 3.5 (hence, subextensive entropy), which opens new perspectives for GRNs inference methods based on information theory and for investigation of the nonextensivity of such networks. The inference algorithm and criterion function proposed here were implemented and included in the DimReduction software, which is freely available at http://sourceforge.net/projects/dimreduction and http://code.google.com/p/dimreduction/.

Construct and Compare Gene Coexpression Networks with DAPfinder and DAPview

Background: DAPfinder and DAPview are novel BRB-ArrayTools plug-ins to construct gene coexpression networks and identify significant differences in pairwise gene-gene coexpression between two phenotypes. Results: Each significant difference in gene-gene association represents a Differentially Associated Pair (DAP). Our tools include several choices of filtering methods, gene-gene association metrics, statistical testing methods and multiple comparison adjustments. Network results are easily displayed in Cytoscape. Analyses of glioma experiments and microarray simulations demonstrate the utility of these tools. Conclusions: DAPfinder is a new friendly-user tool for reconstruction and comparison of biological networks.

Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly in patients with heart failure

Background -: Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly and Thr164Ile were suggested to have an effect in heart failure. We evaluated these polymorphisms relative to clinical characteristics and prognosis of alarge cohort of patients with heart failure of different etiologies. Methods -: We studied 501 patients with heart failure of different etiologies. Mean age was 58 years (standard deviation 14.4 years), 298 (60%) were men. Polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism. Results -: During the mean follow-up of 12.6 months (standard deviation 10.3 months), 188 (38%) patients died. Distribution of genotypes of polymorphism Arg16Gly was different relative to body mass index (chi(2) = 9.797; p = 0.04). Overall the probability of survival was not significantly predicted by genotypes of Gln27Glu, Arg16Gly, or Thr164Ile. Allele and haplotype analysis also did not disclose any significant difference regarding mortality. Exploratory analysis through classification trees pointed towards a potential association between the Gln27Glu polymorphism and mortality in older individuals. Conclusion -: In this study sample, we were not able to demonstrate an overall influence of polymorphisms Gln27Glu and Arg16Gly of beta-2 receptor gene on prognosis. Nevertheless, Gln27Glu polymorphism may have a potential predictive value in older individuals.

The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

Background: NADPH-cytochrome- P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling. Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.

Interaction between polymorphisms of the Human Leukocyte Antigen and HPV-16 Variants on the risk of invasive cervical cancer

Background: Persistent infection with oncogenic types of human papillomavirus (HPV) is the major risk factor for invasive cervical cancer (ICC), and non-European variants of HPV-16 are associated with an increased risk of persistence and ICC. HLA class II polymorphisms are also associated with genetic susceptibility to ICC. Our aim is to verify if these associations are influenced by HPV-16 variability. Methods: We characterized HPV-16 variants by PCR in 107 ICC cases, which were typed for HLA-DQA1, DRB1 and DQB1 genes and compared to 257 controls. We measured the magnitude of associations by logistic regression analysis. Results: European ( E), Asian-American ( AA) and African (Af) variants were identified. Here we show that inverse association between DQB1*05 ( adjusted odds ratio [ OR] = 0.66; 95% confidence interval [CI]: 0.39-1.12]) and HPV-16 positive ICC in our previous report was mostly attributable to AA variant carriers ( OR = 0.27; 95% CI: 0.10-0.75). We observed similar proportions of HLA DRB1*1302 carriers in E-P positive cases and controls, but interestingly, this allele was not found in AA cases ( p = 0.03, Fisher exact test). A positive association with DRB1*15 was observed in both groups of women harboring either E ( OR = 2.99; 95% CI: 1.13-7.86) or AA variants ( OR = 2.34; 95% CI: 1.00-5.46). There was an inverse association between DRB1*04 and ICC among women with HPV-16 carrying the 350T [83L] single nucleotide polymorphism in the E6 gene ( OR = 0.27; 95% CI: 0.08-0.96). An inverse association between DQB1*05 and cases carrying 350G (83V) variants was also found ( OR = 0.37; 95% CI: 0.15-0.89). Conclusion: Our results suggest that the association between HLA polymorphism and risk of ICC might be influenced by the distribution of HPV-16 variants.

A Complete Skull of an Early Cretaceous Sauropod and the Evolution of Advanced Titanosaurians

Advanced titanosaurian sauropods, such as nemegtosaurids and saltasaurids, were diverse and one of the most important groups of herbivores in the terrestrial biotas of the Late Cretaceous. However, little is known about their rise and diversification prior to the Late Cretaceous. Furthermore, the evolution of their highly-modified skull anatomy has been largely hindered by the scarcity of well-preserved cranial remains. A new sauropod dinosaur from the Early Cretaceous of Brazil represents the earliest advanced titanosaurian known to date, demonstrating that the initial diversification of advanced titanosaurians was well under way at least 30 million years before their known radiation in the latest Cretaceous. The new taxon also preserves the most complete skull among titanosaurians, further revealing that their low and elongated diplodocid-like skull morphology appeared much earlier than previously thought.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

P>During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.