Gastrotricha: A Marine Sister for a Freshwater Puzzle

Background: Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data. Methodology/Principal Findings: Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera. Conclusions/Significance: A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.

Double-Stranded RNA-Activated Protein Kinase Is a Key Modulator of Insulin Sensitivity in Physiological Conditions and in Obesity in Mice

The molecular integration of nutrient-and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of kappa B kinase beta. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of kappa B kinase beta phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity. (Endocrinology 153:5261-5274, 2012)

Levels of Selenomonas species in generalized aggressive periodontitis

Goncalves LFH, Fermiano D, Feres M, Figueiredo LC, Teles FRP, Mayer MPA, Faveri M. Levels of Selenomonas species in generalized aggressive periodontitis. J Periodont Res 2012; 47: 711718. (c) 2012 John Wiley & Sons A/S Background and Objective: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. Material and Methods: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the MannWhitney U-test. Results: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects (p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P.gingivalis and S.sputigena were higher in deep sites (probing depth = 5 mm) than in shallow sites (probing depth = 3 mm) (p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of = 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P.gingivalis (r = 0.77; p < 0.01), S.sputigena (r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) (r = 0.42, p < 0.05). Conclusion: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further.

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EPTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the fast multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of beta-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

SMALL INTERFERING RNA TARGETING FOCAL ADHESION KINASE PREVENTS CARDIAC DYSFUNCTION IN ENDOTOXEMIA

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Decreased RNA expression of interleukin 17A in skin of leprosy

Interleukin-17A (IL-17A) is a proinflamatory cytokine that plays an important role in fighting pathogens at mucosal interfaces, by summoning neutrophils and upregulating cytoplasmatic antimicrobial peptides. So far, the presence of IL-17A in leprosy has not been demonstrated. The expression of IL-17A and related cytokines (IL-6 and IL-23p19) was addressed through RNA extraction and cDNA quantitative amplification in macerated biopsies of active lesions of 48 leprosy patients and 20 fragments of normal skin of individuals. Blood levels of IL-17A, IL-23p19 and IL-6 were determined by ELISA. We found an abrogated mRNA IL-17A response in all biopsies of leprosy patients, as compared with controls. Circulating IL-17A and IL-23p19 were undetectable in both patients and controls, but IL-6 was higher in lepromatous patients. Although at low levels, IL-17A mRNA in lepromatous patients had an inverse linear correlation with bacillary burden. Low expression of IL-17A in patients may be a constitutive genetic feature of leprosy patients or a circumstantial event induced by the local presence of the pathogen, as an escape mechanism.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas

Background: Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. Objective: The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. Methods: Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. Results: Computer-generated genomic analysis tools identified 13 722 and 14 993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. Conclusion: We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

Exploring the Schistosoma mansoni adult male transcriptome using RNA-seq

Schistosoma mansoni is one of the agents of schistosomiasis, a chronic and debilitating disease. Here we, present a transcriptome-wide characterization of adult S. mansoni males by high-throughput RNA-sequencing. We obtained 1,620,432 high-quality ESTs from a directional strand-specific cDNA library, resulting in a 26% higher coverage of genome bases than that of the public ESTs available at NCBI. With a 15 x-deep coverage of transcribed genomic regions, our data were able to (i) confirm for the first time 990 predictions without previous evidence of transcription; (ii) correct gene predictions; (iii) discover 989 and 1196 RNA-seq contigs that map to intergenic and intronic genomic regions, respectively, where no gene had been predicted before. These contigs could represent new protein-coding genes or non-coding RNAs (ncRNAs). Interestingly, we identified 11 novel Micro-exon genes (MEGs). These data reveal new features of the S. mansoni transcriptional landscape and significantly advance our understanding of the parasite transcriptome. (c) 2011 Elsevier Inc. All rights reserved.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models. (C) 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

Trypanosoma (Megatrypanum) melophagium in the Sheep Ked Melophagus ovinus from Organic Farms in Croatia: Phylogenetic Inferences Support Restriction to Sheep and Sheep Keds and Close Relationship with Trypanosomes from Other Ruminant Species

Trypanosoma (Megatrypanum) melophagium is a parasite of sheep transmitted by sheep keds, the sheep-restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae). Sheep keds were 100% prevalent in sheep from five organic farms in Croatia, Southeastern Europe, whereas trypanosomes morphologically compatible with T. melophagium were 86% prevalent in the guts of the sheep keds. Multilocus phylogenetic analyses using sequences of small subunit rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase, spliced leader, and internal transcribed spacer 1 of the rDNA distinguished T. melophagium from all allied trypanosomes from other ruminant species and placed the trypanosome in the subgenus Megatrypanum. Trypanosomes from sheep keds from Croatia and Scotland, the only available isolates for comparison, shared identical sequences. All biologic and phylogenetic inferences support the restriction of T. melophagium to sheep and, especially, to the sheep keds. The comparison of trypanosomes from sheep, cattle, and deer from the same country, which was never achieved before this work, strongly supported the host-restricted specificity of trypanosomes of the subgenus Megatrypanum. Our findings indicate that with the expansion of organic farms, both sheep keds and T. melophagium may re-emerge as parasitic infections of sheep.

Cwc24p Is a General Saccharomyces cerevisiae Splicing Factor Required for the Stable U2 snRNP Binding to Primary Transcripts

Splicing of primary transcripts is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamics of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to primary transcripts, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for stabilizing the U2 association with primary transcripts, and therefore, especially important for splicing of RNAs containing non- consensus branchpoint sequences.

Parasites reveal movement of bats between the New and Old Worlds

The global distribution of bat taxa indicates that the Atlantic and Pacific Oceans are effective barriers to movement between the Old and New Worlds. For instance, one of the major suborders, Yinpterochiroptera, has an exclusively Old World distribution, and within the other, Yangochiroptera, no species and only five genera are common to both. However, as bats are sometimes blown out to sea, and have colonised isolated islands, occasional natural movement between the New and Old Worlds does appear to be possible. Here we identify new genotypes of a blood parasite, Trypanosoma dionisii, in Old World bats that are closely related to South American strains. Using highly conservative calibration points, divergence of Old and New World strains is estimated to have occurred 3.2-5.0 million years ago (MYA), depending on the method used (upper 95% CL for maximum time 11.4 MYA). The true date of divergence is likely to be considerably more recent. These results demonstrate that taxon-specific parasites can indicate historical movements of their hosts, even where their hosts may have left no lasting phylogenetic footprint. (C) 2012 Elsevier Inc. All rights reserved.