A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

Combining Partial True Discovery Guarantee Procedures.

Closed testing has recently been shown to be optimal for simultaneous true discovery proportion control. It is, however, challenging to construct true discovery guarantee procedures in such a way that it focuses power on some feature sets chosen by users based on their specific interest or expertise. We propose a procedure that allows users to target power on prespecified feature sets, that is, "focus sets." Still, the method also allows inference for feature sets chosen post hoc, that is, "nonfocus sets," for which we deduce a true discovery lower confidence bound by interpolation. Our procedure is built from partial true discovery guarantee procedures combined with Holm's procedure and is a conservative shortcut to the closed testing procedure. A simulation study confirms that the statistical power of our method is relatively high for focus sets, at the cost of power for nonfocus sets, as desired. In addition, we investigate its power property for sets with specific structures, for example, trees and directed acyclic graphs. We also compare our method with AdaFilter in the context of replicability analysis. The application of our method is illustrated with a gene ontology analysis in gene expression data.

Closed testing with Globaltest, with application in metabolomics.

The Globaltest is a powerful test for the global null hypothesis that there is no association between a group of features and a response of interest, which is popular in pathway testing in metabolomics. Evaluating multiple feature sets, however, requires multiple testing correction. In this paper, we propose a multiple testing method, based on closed testing, specifically designed for the Globaltest. The proposed method controls the familywise error rate simultaneously over all possible feature sets, and therefore allows post hoc inference, that is, the researcher may choose feature sets of interest after seeing the data without jeopardizing error control. To circumvent the exponential computation time of closed testing, we derive a novel shortcut that allows exact closed testing to be performed on the scale of metabolomics data. An R package ctgt is available on comprehensive R archive network for the implementation of the shortcut procedure, with applications on several real metabolomics data examples.

The Product of the Fission Yeast fhl1 Gene Binds to the HomolE Box and Activates In Vitro Transcription of Ribosomal Protein Genes.

Fission yeast ribosomal protein genes (RPGs) contain a HomolD box as a core promoter element required for transcription. Some of the RPGs also contain a consensus sequence named HomolE, located upstream of the HomolD box. The HomolE box acts as an upstream activating sequence (UAS), and it is able to activate transcription in RPG promoters containing a HomolD box. In this work, we identified a HomolE-binding protein (HEBP) as a polypeptide of 100 kDa, which was able to bind to the HomolE box in a Southwestern blot assay. The features of this polypeptide were similar to the product of the fhl1 gene of fission yeast. The Fhl1 protein is the homolog of the FHL1 protein of budding yeast and possesses fork-head-associated (FHA) and fork-head (FH) domains. The product of the fhl1 gene was expressed and purified from bacteria, and it was demonstrated that is able to bind the HomolE box in an electrophoretic mobility assay (EMSA), as well as being able to activate in vitro transcription from an RPG gene promoter containing HomolE boxes upstream of the HomolD box. These results indicate that the product of the fhl1 gene of fission yeast can bind to the HomolE box, and it activates the transcription of RPGs.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

The Catalytic Subunit of Schizosaccharomyces pombe CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4).

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well.

Globaltest confidence regions and their application to ridge regression.

We construct confidence regions in high dimensions by inverting the globaltest statistics, and use them to choose the tuning parameter for penalized regression. The selected model corresponds to the point in the confidence region of the parameters that minimizes the penalty, making it the least complex model that still has acceptable fit according to the test that defines the confidence region. As the globaltest is particularly powerful in the presence of many weak predictors, it connects well to ridge regression, and we thus focus on ridge penalties in this paper. The confidence region method is quick to calculate, intuitive, and gives decent predictive potential. As a tuning parameter selection method it may even outperform classical methods such as cross-validation in terms of mean squared error of prediction, especially when the signal is weak. We illustrate the method for linear models in simulation study and for Cox models in real gene expression data of breast cancer samples.

Prevalence, infected density or individual probability of infection? Assessing vector infection risk in the wild transmission of Chagas disease.

Vector-borne infectious disease dynamics result mainly from the intertwined effect of the diversity, abundance, and behaviour of hosts and vectors. Most studies, however, have analysed the relationship between host-species diversity and infection risk, focusing on vector population instead of individuals, probably dismissing the level at which the transmission process occurs. In this paper, we examine the importance of the host community in accounting for infection risk, at both population and individual levels, using the wild transmission of the protozoan that causes Chagas disease as a vector-borne disease model. Chagas disease is caused by Trypanosoma cruzi, transmitted by triatomine insects to mammals. We assessed if T. cruzi infection in vectors is explained by small mammal diversity and their densities (total and infected), when infection risk is measured at population level as infection prevalence (under a frequency-dependent transmission approach) and as density of infected vectors (density-dependent transmission approach), and when measured at individual level as vector infection probability. We analysed the infection status of 1974 vectors and co-occurring small mammal hosts in a semiarid-Mediterranean ecosystem. Results revealed that regardless of the level of analysis, only one host rodent species accounted for most variation in vector infection risk, suggesting a key role in the transmission cycle. To determine the factors explaining vector-borne disease dynamics, infection risk should be assessed at different scales, reflecting the factors meaningful from the vector's perspective and considering vector class-specific features.

Organs infected with Trypanosoma cruzi and DTU identification in the naturally infected rodent Octodon degus.

Chagas disease is a public health problem in America. Its parasite, Trypanosoma cruzi, presents different discrete typing units (DTUs), colonizes organs of mammalian hosts in chronic infections, and presents tropism for particular organs in experimental infections. We evaluated T. cruzi tropism towards organs on the naturally infected rodent Octodon degus, identifying the parasites' DTUs, by means of conventional PCR and hybridization. Almost all the analyzed organs presented T. cruzi. More than 42% of the tested oesophagus, skin, skeletal muscle, brain and intestine showed T. cruzi DNA. Other nine types of organs were infected in over 15%. These results suggest that there is some tropism by T. cruzi in chronically infected O. degus. DTU TcV was present in 92.5% of infected organs with identified DTUs; this DTU is frequently reported in human infections in the Southern Cone of South America. Few organs showed mixed DTU infections. This is one of the few reports on the outcome of chronic natural T. cruzi-infection in wild mammal hosts exposed to naturally infected vectors.

All-Resolutions Inference for brain imaging.

The most prevalent approach to activation localization in neuroimaging is to identify brain regions as contiguous supra-threshold clusters, check their significance using random field theory, and correct for the multiple clusters being tested. Besides recent criticism on the validity of the random field assumption, a spatial specificity paradox remains: the larger the detected cluster, the less we know about the location of activation within that cluster. This is because cluster inference implies "there exists at least one voxel with an evoked response in the cluster", and not that "all the voxels in the cluster have an evoked response". Inference on voxels within selected clusters is considered bad practice, due to the voxel-wise false positive rate inflation associated with this circular inference. Here, we propose a remedy to the spatial specificity paradox. By applying recent results from the multiple testing statistical literature, we are able to quantify the proportion of truly active voxels within selected clusters, an approach we call All-Resolutions Inference (ARI). If this proportion is high, the paradox vanishes. If it is low, we can further "drill down" from the cluster level to sub-regions, and even to individual voxels, in order to pinpoint the origin of the activation. In fact, ARI allows inference on the proportion of activation in all voxel sets, no matter how large or small, however these have been selected, all from the same data. We use two fMRI datasets to demonstrate the non-triviality of the spatial specificity paradox, and its resolution using ARI. We verify that the endless circularity permitted by ARI does not render its estimates overly conservative using both simulation, and a data split.

Minimally adaptive BH: A tiny but uniform improvement of the procedure of Benjamini and Hochberg.

We define an adaptive procedure for control of the false discovery rate that is uniformly more powerful than the procedure of Benjamini and Hochberg. The power gain is tiny, however, and only appreciable for small numbers of hypotheses. We illustrate the new method with the case of two hypotheses, for which so far no procedure was known that controls false discovery rate but not also familywise error rate under positive dependence.

Spatial distribution of an infectious disease in a small mammal community.

Chagas disease is a zoonosis caused by the parasite Trypanosoma cruzi and transmitted by insect vectors to several mammals, but little is known about its spatial epidemiology. We assessed the spatial distribution of T. cruzi infection in vectors and small mammals to test if mammal infection status is related to the proximity to vector colonies. During four consecutive years we captured and georeferenced the locations of mammal species and colonies of Mepraia spinolai, a restricted-movement vector. Infection status on mammals and vectors was evaluated by molecular techniques. To examine the effect of vector colonies on mammal infection status, we constructed an infection distance index using the distance between the location of each captured mammal to each vector colony and the average T. cruzi prevalence of each vector colony, weighted by the number of colonies assessed. We collected and evaluated T. cruzi infection in 944 mammals and 1976 M. spinolai. We found a significant effect of the infection distance index in explaining their infection status, when considering all mammal species together. By examining the most abundant species separately, we found this effect only for the diurnal and gregarious rodent Octodon degus. Spatially explicit models involving the prevalence and location of infected vectors and hosts had not been reported previously for a wild disease.

Trypanosoma cruzi burden, genotypes, and clinical evaluation of Chilean patients with chronic Chagas cardiopathy.

There are currently no biomarkers to assess which patients with chronic indeterminate Chagas disease will develop heart disease and which will spend their entire life in this state. We hypothetize that the parasite burden and Trypanosoma cruzi genotypes are related to the presence of heart disease in patients with Chagas disease. This study is aimed to investigate the parasite burden and T. cruzi genotypes in chagasic cardiopaths versus chagasic individuals without cardiac involvement according to the New York Heart Association. Patients with chronic Chagas disease, 50 with and 50 without cardiopathy (controls), groups A and B, respectively, were submitted to anamnesis, physical examination, and electrocardiogram. Echo-Doppler was performed for group A; all important known causes of cardiopathy were discarded. Xenodiagnosis, conventional PCR, and quantitative PCR were performed on patients of both groups. T. cruzi genotyping was done for 25 patients of group A and 20 of group B. The 50 cardiopaths had 80 electrocardiographic alterations, most of them in grade II of the New York Heart Association classification; 49 were classified in grade I by Echo-Doppler, and only one patient was in grade III. The difference in average parasitemia in patients of groups A and B was not significant. The most frequent T. cruzi DTU found was TcV. The parasite burden and genotype of the groups with and without cardiopathy were similar. Graphical abstract Imagen 1 Chronic chagas cardiopathy chest X-ray heart enlargement Figure 2 Chronic Chagas cardiopathy microaneurism of left ventricle. Cineangiography.

Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

Phylogenetic incongruence inferred with two mitochondrial genes in Mepraia spp. and Triatoma eratyrusiformis(Hemiptera, Reduviidae).

Mitochondrial DNA (mtDNA) is widely used to clarify phylogenetic relationships among and within species, and to determine population structure. Due to the linked nature of mtDNA genes it is expected that different genes will show similar results. Phylogenetic incongruence using mtDNA genes may result from processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, contamination, and recombination. In this study we used sequences from two mitochondrial genes (cytochrome b and cytochrome oxidase subunit I) from the wild vectors of Chagas disease, Triatoma eratyrusiformis and Mepraia species to test for topological congruence. The results showed some cases of phylogenetic incongruence due to misplacement of four haplotypes of four individuals. We discuss the possible causes of such incongruence and suggest that the explanation is an intra-individual variation likely due to heteroplasmy. This phenomenon is an independent evidence of common ancestry between these taxa.

Rotation-based multiple testing in the multivariate linear model.

In observational microarray studies, issues of confounding invariably arise. One approach to account for measured confounders is to include them as covariates in a multivariate linear model. For this model, however, the application of permutation-based multiple testing procedures is problematic because exchangeability of responses, in general, does not hold. Nevertheless, it is possible to achieve rotatability of transformed responses at the cost of a distributional assumption. We argue that rotation-based multiple testing, by allowing for adjustments for confounding, represents an important extension of permutation-based multiple testing procedures. The proposed methodology is illustrated with a microarray observational study on breast cancer tumors. Software to perform the procedure described in this article is available in the flip R package.

Multiple hypothesis testing in genomics.

This paper presents an overview of the current state of the art in multiple testing in genomics data from a user's perspective. We describe methods for familywise error control, false discovery rate control and false discovery proportion estimation and confidence, both conceptually and practically, and explain when to use which type of error rate. We elaborate on the assumptions underlying the methods and discuss pitfalls in the interpretation of results. In our discussion, we take into account the exploratory nature of genomics experiments, looking at selection of genes before or after testing, and at the role of validation experiments.

In Vitro Identification of Phosphorylation Sites on TcPolß by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolß in Trypanosoma cruzi Epimastigotes.

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolß) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolß by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolß. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolß. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolß phosphorylation and enzymatic activity in T. cruzi epimastigotes.

Evaluation of nifurtimox treatment of chronic Chagas disease by means of several parasitological methods.

Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples.

Aging Hallmarks and the Role of Oxidative Stress.

Aging is a complex biological process accompanied by a progressive decline in the physical function of the organism and an increased risk of age-related chronic diseases such as cardiovascular diseases, cancer, and neurodegenerative diseases. Studies have established that there exist nine hallmarks of the aging process, including (i) telomere shortening, (ii) genomic instability, (iii) epigenetic modifications, (iv) mitochondrial dysfunction, (v) loss of proteostasis, (vi) dysregulated nutrient sensing, (vii) stem cell exhaustion, (viii) cellular senescence, and (ix) altered cellular communication. All these alterations have been linked to sustained systemic inflammation, and these mechanisms contribute to the aging process in timing not clearly determined yet. Nevertheless, mitochondrial dysfunction is one of the most important mechanisms contributing to the aging process. Mitochondria is the primary endogenous source of reactive oxygen species (ROS). During the aging process, there is a decline in ATP production and elevated ROS production together with a decline in the antioxidant defense. Elevated ROS levels can cause oxidative stress and severe damage to the cell, organelle membranes, DNA, lipids, and proteins. This damage contributes to the aging phenotype. In this review, we summarize recent advances in the mechanisms of aging with an emphasis on mitochondrial dysfunction and ROS production.