Genome assembly and phylogenomic data analyses using plastid data: Contrasting species tree estimation methods.

Phylogenomics has become increasingly popular in recent years mostly due to the increased affordability of next generation sequencing techniques. Phylogenomics has sparked interest in multiple fields of research, including systematics, ecology, epidemiology, and even personalized medicine, agriculture and pharmacy. Despite this trend, it is usually difficult to learn and understand how the analyses were done, how the results were obtained, and most importantly, how to replicate the study. Here we present the data and all of the code utilized to perform phylogenomic inferences using plastome data: from raw data to extensive phylogenetic inference and accuracy assessment. The data presented here utilizes plastome sequences available on GenBank (accession numbers of 94 species are available below) and the code is also available at https://github.com/deisejpg/rosids. Gonçalves et al. is the research article associated with the data analyses presented here.

Cardiovascular assessment in Female Spotted Paca ( Cuniculus paca ) / Avaliação cardiovascular em fêmea da Cúciculus paca

Aiming to provide cardiovascular morphophysiology information on the Cuniculus paca, an important neotropical rodent, eight healthy adult females of this species were evaluated three times by echocardiography under general anesthesia with isoflurane every 15 days. The exams were performed by a single experienced evaluator with the animals positioned in right and left decubitus. Posteriorly, two expert evaluators measured the cardiac chambers, walls and flow patterns, by B-mode, M-mode, and Doppler ultrasonography. The resulting values were compared among evaluators and periods by the Bland-Altman agreement test and several descriptive statistics were presented for each parameter. Echocardiographic images were obtained between the second and fifth left and right intercostal spaces, enabling the measurement of heart chambers and walls, mitral, tricuspid, aortic and pulmonary valves blood flows, and the ejection and shortening fractions calculation. None of the studied variables showed inter-observers or inter-periods variations. This study provided some normal echocardiographic variables, applicable to epidemiological, pathophysiological or case studies in the Cuniculus paca and phylogenetically close species.(AU)

Com o objetivo de fornecer informações da morfofisiologia cardiovascular da Cuniculus paca, um importante roedor neotropical, oito fêmeas adultas saudáveis dessa espécie foram avaliadas três vezes pela ecocardiografia, sob anestesia geral com isoflurano, a cada 15 dias. Os exames foram realizados por um único avaliador experiente, com os animais posicionados em decúbito direito e esquerdo. Posteriormente, dois avaliadores experientes mediram as câmaras cardíacas, as paredes e os padrões de fluxo, por meio do modo B, do modo M e da ultrassonografia Doppler. Os valores resultantes foram comparados entre avaliadores e períodos pelo teste de concordância de Bland-Altman, e várias estatísticas descritivas foram apresentadas para cada parâmetro. As imagens ecocardiográficas foram obtidas entre o segundo e o quinto espaços intercostais esquerdo e direito, o que permitiu a medição das câmaras e paredes cardíacas, do fluxo sanguíneo pelas válvulas mitral, tricúspide, aórtica e pulmonar e o cálculo das frações de ejeção e encurtamento. Nenhuma das variáveis estudadas mostrou variações entre os avaliadores ou entre os períodos. Este estudo fornece algumas variáveis ecocardiográficas normais, aplicáveis a estudos epidemiológicos, fisiopatológicos ou de casos na Cuniculus paca e em espécies filogeneticamente próximas.(AU)

Esclerosis tuberosa. A propósito de un caso / Tuberous sclerosis: based on one case

Dentro de los síndromes neurocutáneos o facomatosis, en la primera infancia; la asociación de máculas acrómicas, convulsiones y retraso del desarrollo psicomotor es altamente sugerente de esclerosis tuberosa (ET). Se presenta el caso de un varón en el cual, precozmente, a los cuatro meses de edad, se realiza este diagnóstico. Se discute el enfoque diagnóstico a la luz de los conocimientos actuales sobre dicha entidad, según la literatura médica (AU)

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Esclerosis tuberosa: a propósito de un caso / Tuberous sclerosis: a case purpose

Dentro de los síndromes neurocutáneos o facomatosis, en la primera infancia: la asociación de máculas acrómicas, convulsiones y retraso del desarrollo psicomotor es altamente sugerente de esclerosis tuberosa (ET). Se presenta el caso de un varón en el cual, precozmente, a los cuatro meses de edad, se realiza este diagnóstico. Se discute el enfoque diagnóstico a la luz de los conocimientos actuales sobre dicha entidad, según la leteratura médica(AU)

Tumor necrosis factor alpha triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation.

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha contributes to the development of acute pancreatitis. Because TNF-alpha is involved in the control of apoptosis, we studied its interaction with the pancreatic apoptotic pathway. METHODS: Pancreatic acinar AR4-2J cells were used. Apoptosis was monitored by morphologic and biochemical criteria. RESULTS: TNF-alpha induced apoptosis in AR4-2J cells. Induction was strongly enhanced in cells treated with actinomycin D, suggesting that TNF-alpha activated concomitantly an antiapoptotic mechanism through newly synthesized proteins. This mechanism involved activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases because their inhibition worsened TNF-alpha-induced apoptosis. The antiapoptotic pancreatitis-associated protein (PAP) I is a candidate for mediating TNF-alpha activity. Its expression is induced by TNF-alpha, and cells overexpressing PAP I show significantly less apoptosis on exposure to TNF-alpha. We examined whether TNF-alpha induction of PAP I expression was mediated by NF-kappaB or MAP kinases by using specific inhibitors of both pathways. Inhibition of NF-kappaB had no effect. However, inhibitors of MEK1 eliminated PAP I induction. CONCLUSIONS: TNF-alpha induces concomitantly proapoptotic and antiapoptotic mechanisms in pancreatic AR4-2J cells. Antiapoptotic mechanisms are mediated by NF-kappaB and MAP kinases, and PAP I is one of the effectors of apoptosis inhibition.

Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter.

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5' flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPalpha or C/EBPbeta expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPalpha and C/EBPbeta. Co-transfection with C/EBPalpha or C/EBPbeta expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPalpha or C/EBPbeta still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPalpha and C/EBPbeta trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.

The pancreatitis-associated protein is induced by free radicals in AR4-2J cells and confers cell resistance to apoptosis.

BACKGROUND & AIMS: Free radicals are involved in the pathogenesis of acute pancreatitis, during which pancreatitis-associated protein (PAP)-I is overexpressed. We explored whether PAP-I expression could be induced by oxidative stress and whether it could affect apoptosis. METHODS: AR4-2J cells were exposed to H2O2 or menadione, and PAP-I messenger RNA (mRNA) expression was analyzed by Northern blotting. RESULTS: Maximal expression was observed with 0.1 mmol/L H2O2 or with 0.05 mmol/L menadione. Induction was detectable after 12 hours, reached a climax at 18 hours, and then decreased. Pretreatment of the cells with pyrrolidine dithiocarbamate completely abolished PAP-I mRNA induction, suggesting involvement of NFkappaB in the signaling pathway. These findings were confirmed in transient transfection assays using a plasmid containing the PAP-I promoter linked to the chloramphenicol acetyltransferase reporter gene. Then the relationship between PAP-I induction and protection against cell damage during oxidative stress was considered. Constitutive PAP-I expression in AR4-2J cells after transfection with PAP-I complementary DNA conferred significant resistance to apoptosis induced by low doses of H2O2 but not to necrosis induced by high doses of H2O2. CONCLUSIONS: These results suggest that during oxidative stress, PAP-I might be part of a mechanism of pancreatic cell protection against apoptosis.

Characterization of a silencer regulatory element in the rat PAP I gene which confers tissue-specific expression and is promoter-dependent.

Previous analysis of the rat PAP I promoter indicated that the region between nt -180 and -81 possessed silencer activity in cells that did not express PAP I. Based on this finding, we performed a series of experiments to characterize functionally that region and analyze the nuclear proteins interacting with it. Transient transfection assays were conducted in the fibroblast Rat2 cell line, in which PAP I is not expressed, and in the pancreatic cell line AR-42J, expressing PAP I, using the CAT gene as reporter. Experiments in Rat2 cells revealed that the sequence with silencer activity was located within the rep27 region (position -180/-153). Suppressor activity was observed when rep27 was inserted upstream from the core PAP I promoter, in both orientations. By contrast, inserting the rep27 region in front of the promoters of SV40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-dependent. In pancreatic AR-42J cells, rep27 act as a positive element but did not alter CAT expression when inserted in front of the core PAP I promoter or heterologous promoters. Electrophoretic mobility shift assays allowed identification of specific DNA-protein complexes. The shifted complex migrated at the same position with both Rat2 and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with rat nuclear extracts from healthy pancreas, pancreas with acute pancreatitis, liver, kidney, spleen, and small intestine. Results suggest that the rep27 cis-acting element contributes to the tissue specific expression of the PAP I gene. That activity could be mediated by the synergistic action of several transcription factors, one of which being present in all cells.

The pancreatitis-associated protein I promoter allows targeting to the pancreas of a foreign gene, whose expression is up-regulated during pancreatic inflammation.

The pancreatitis-associated protein I (PAP I) is a pancreatic secretory protein expressed in pancreas during acute pancreatitis but not in the healthy pancreas. The promoter of the PAP I gene thus represents a potential candidate to drive expression of therapeutic molecules to the diseased pancreas. In this work, we have constructed recombinant adenoviruses harboring the chloramphenicol acetyltransferase (CAT) gene driven by several fragments of the PAP I promoter and have characterized their properties in vitro and in vivo. In vitro studies showed that the transduction of the pancreatic cell line AR-42J with these adenoviruses led to low levels of CAT activity in basal conditions. After stimulation with a combination of interleukin-6 and dexamethasone or after induction of oxidative stress, CAT activity was strongly induced, a characteristic of the endogenous PAP I gene. Stimulation was maximal when constructs comprised 1253 base pairs of the PAP I promoter, upstream from initiation of transcription, and decreased with shorter fragments of 317, 180, 118 or 61 base pairs. The recombinant adenovirus containing the CAT gene under the control of the PAP I promoter fragment (-1253/+10) was also tested in vivo. Following administration by intravenous injection into mice, CAT activity was measured in several tissues 96 h later. In healthy animals, low but significant CAT activity was detected in pancreas, compared with near background values observed in the other tissues. When experimental acute pancreatitis was induced, CAT expression was strongly enhanced only in pancreas. In control experiments with adenoviruses in which the CAT gene was driven by the cytomegalovirus promoter, higher levels of expression were observed in all tissues. Expression was not modified after induction of acute pancreatitis. In conclusion, this study shows that (i) a recombinant adenovirus containing a fragment of the PAP I promoter allows specific targeting of a reporter gene to the mouse pancreas and (ii) expression of the reporter gene in pancreas is induced during acute pancreatitis. Adenovirus-mediated gene therapy of acute pancreatitis is therefore conceivable.

Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth.

To characterize at the molecular level the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis, we have developed a strategy in which the phenotype of the pancreatitis affected pancreas is established by characterization of a large number of its transcripts. Herein, we describe the cloning, sequence, and expression of a new gene, named p8, which is strongly activated in pancreatic acinar cells during the acute phase of pancreatitis, in developing pancreas and during pancreatic regeneration. In acinar cells, p8 mRNA is expressed rapidly and specifically in response to cellular pancreatitis-induced injury; its induction occurred almost similarly in edematous and necrohemorrhagic pancreatitis, indicating that p8 mRNA is maximally activated even in response to a mild pancreatic injury. Furthermore, in vitro studies suggest that p8 mRNA is induced in pancreatic and non-pancreatic cells in response to some apoptotic stimuli. p8 acts as a promoter of cellular growth factor when its cDNA is transfected into COS-7 and AR4-2J cells. Although we failed to identify p8-related sequences, analysis of its primary and secondary structure suggests that p8 is a basic helix-turn-helix-containing gene with slight homology to several homeotic genes and sufficient signal to be targeted to the nucleus. We therefore propose p8 as a putative transcriptional factor which can regulate pancreatic growth.

Mechanism of PAP I gene induction during hepatocarcinogenesis: clinical implications.

Pancreatitis-associated protein I (PAP I) is a secretory protein first described as an acute phase reactant during acute pancreatitis. Recently, induction of the PAP I gene was also described in liver during hepatocarcinogenesis. To investigate the molecular mechanisms of this induction, we used constructs carrying progressive deletions of the PAP I promoter fused to the CAT gene. We showed that the silencer conferring tissue specificity on the PAP I gene was inactive in hepatoma cells. Then, in an vitro transcription system, we compared the transcription capacity of nuclear extracts from normal liver and HepG2 cells on constructs containing the silencer. The results confirmed that a trans-acting factor interacting with the PAP I silencer was present in liver cells and absent from hepatoma cells. On the other hand, immunohistochemistry showed that PAP I was expressed in a limited number of transformed hepatocytes. It was concluded that expression of PAP I in hepatocarcinoma occurred through inactivation of its silencer element and was not concomitant in all malignant cells. On that basis, we assayed PAP I in serum from patients with chronic hepatitis, liver cirrhosis or hepatocarcinoma. PAP I levels were normal in chronic active or persistent hepatitis, significantly higher in cirrhosis and strongly elevated in hepatocarcinoma. Because those clinical entities often develop in that sequence, serum PAP I appeared as a potential marker of hepatocarcinoma development.

Induction of lithostathine/reg mRNA expression by serum from rats with acute pancreatitis and cytokines in pancreatic acinar AR-42J cells.

During the acute phase of pancreatitis, expression of most pancreatic enzymes decreases, whereas mRNAs of pancreatitis associated protein and lithostathine/reg increase dramatically. In the present study we have investigated the effect of serum from rats with acute pancreatitis (SAP) and cytokines on the lithostathine/reg mRNA expression in AR-42J cells. Lithostathine/reg mRNA was strongly induced by SAP in a dose-dependent manner. Induction was abolished by preheating the SAP or by treating the cells with cycloheximide. Treatment with interleukins (IL) IL-1 or IL-6 or dexamethasone alone was ineffective. Combination of IL-1 with IL-6 was also ineffective. Combination of IL-6 with dexamethasone resulted in strong induction of the lithostathine/reg gene, but the further addition of IL-1 to the mixture reduced induction. Treatment with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma) induced lithostathine/reg mRNA expression. Combination of dexamethasone with TNFalpha or IFNgamma showed an inhibitory effect on lithostathine/reg mRNA expression. These findings suggest that expression of the lithostathine/reg mRNA during acute pancreatitis could be mediated by specific combinations of cytokines and/or glucocorticoids.

Scattering by a metallic cylinder on a substrate: burying effects.

Experimental measurements of the light scattered from a metallic circular cylinder, with a section diameter comparable with the incident wavelength, upon a metallic flat substrate are presented. The validity of the theoretical numerical calculation obtained from a one-dimensional model based on Maxwell's coupled integral equations for real metals is demonstrated by comparison with the experimental results. The theoretical model is able to reproduce a geometric feature as the partial burying of the cylinder in the substrate because of the sputtering process.

Identification of a transcriptional regulatory region of the rat pancreatitis-associated protein I (PAP I) gene that confers tissue specificity.

We have previously characterized the rat pancreatitis-associated protein I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated with the DNA sequence comprising nt -1253 to + 10 of the gene. That sequence strongly promoted the transcription of the promotorless chloramphenicol acetyltranferase (CAT) gene in cells of pancreatic origin (AR-42J) but not in cells of non-pancreatic origin (Rat 2 and IEC 6). The influence on CAT expression of stepwise 5' deletions in the promoter sequence was monitored in the three cell lines. In pancreatic AR-42J cells, deletion down to position -926 did not affect significantly the expression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not have any additional effect, but a further deletion to nt -180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 resulted in a further reduction to about one-third of that. Finally, deletion down to nt -61 further reduced activity by a factor of 3, although it remained above background. These results suggest the presence of several positive cis-acting elements in the PAP I promoter. In non-pancreatic cells, CAT expression remained very low when the promoter was deleted down to nt -180. Yet, deletion from -180 to -118 significantly increased CAT expression, suggesting suppression of a negative cis-acting element. Further deletion down to nt -61 decreased CAT activity by a factor of 5. The region between nt -180 and -61 was subjected to footprint analysis. A similar pattern of DNase protection was obtained with AR-42J and Rat 2 nuclear extracts, the only protected region extending from nt -125 to -95. That region was further analysed by inserting the nt -180 to -81 fragment, in both orientations, upstream of thymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but reduced in Rat 2 cells. These results indicated the presence of cell-specific positive and negative elements within that region.

Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region.

Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.