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Defining functional diversity for lignocellulose degradation in a microbial community using multi-omics studies
No full textAbstract\ud
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Background\ud
Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform “community-level” metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes.\ud
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Results\ud
Here, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of Bacteroidetes and Proteobacteria and high relative abundance of genera Asticcacaulis, Leadbetterella and Truepera. The eukaryotic members of the community were enriched in peritrich ciliates from genus Telotrochidium that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of hydrolytic cellulases, hemicellulases and carbohydrate-binding modules involved in lignocellulose degradation. The expression of these activities correlated to the changes in the biomass composition observed by FTIR and ssNMR measurements.\ud
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Conclusions\ud
A combination of mass spectrometry-based proteomics coupled with metatranscriptomics has enabled the identification of a large number of lignocellulose degrading enzymes that can now be further explored for the development of improved enzyme cocktails for the treatment of plant-based feedstocks. In addition to the expected carbohydrate-active enzymes, our studies reveal a large number of unknown proteins, some of which may play a crucial role in community-based lignocellulose degradation.This work was funded by Biotechnology and Biological Sciences Research\ud
Council (BBSRC) Grants BB/1018492/1, BB/K020358/1 and BB/P027717/1, the\ud
BBSRC Network in Biotechnology and Bioenergy BIOCATNET and São Paulo\ud
Research Foundation (FAPESP) Grant 10/52362-5. ERdA thanks EMBRAPA\ud
Instrumentation São Carlos and Dr. Luiz Alberto Colnago for providing the\ud
NMR facility and CNPq Grant 312852/2014-2. The authors would like to thank\ud
Deborah Rathbone and Susan Heywood from the Biorenewables Develop‑\ud
ment Centre for technical assistance in rRNA amplicon sequencing
Amino acid permease 3 (aap3) coding sequence as a target for Leishmania identification and diagnosis of leishmaniases using high resolution melting analysis
No full textAbstract\ud
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Background\ud
The leishmaniases comprise a spectrum of clinical manifestations caused by different species of Leishmania. Identification of species is important for diagnosis, treatment and follow-up management. However, there is no gold standard for species identification. High resolution melting analysis (HRM) offers a possibility to differentiate Leishmania species without the need for processing of the PCR-product. The amino acid permease 3 (aap3) gene is an exclusive target for trypanosomatids and is conserved among Leishmania spp., thus it can be a valuable target for an HRM assay for diagnosis of the leishmaniases.\ud
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Results\ud
The HRM dissociation profiles of three amplicons targeting the aap3-coding region allowed the discrimination of L. (Leishmania) donovani, L. (L.) infantum, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) shawi using DNA from promastigote cultures. The protocol was validated with DNA samples from clinical infection in humans and a cat, naturally infected sand flies, and experimentally infected mice.\ud
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Conclusions\ud
HRM analysis using the aap3 coding sequence as target is a relatively cheap, fast and robust strategy to detect and discriminate Leishmania species from all the endemic regions worldwide. The target and method proved to be useful in clinical, field and experimental samples, thus it could be used as a tool in diagnosis as well as ecological and epidemiological studies.This work was supported by grants from the University of Bergen, Norwegian Centre for International Cooperation in Education (SIU) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - collaboration project CAPES-SIU-2015/10002, and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). None of the funding bodies had any role in the design of the study, data collection, analysis or writing of the manuscript
Low-FODMAP diet in the management of irritable bowel syndrome
No full textAbstract
Background
Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) are short-chain carbohydrates poorly absorbed by humans due to their small size, high osmotic activity, and the speed with which they are fermented by the microbiota. This causes abdominal pain, diarrhea and or constipation, and bloating. Studies about low-FODMAP diet to reduce the symptoms presented by patients with irritable bowel syndrome (IBS) have recently grown. This study aims to identify the characteristics and the risks of low-FODMAP diet to irritable bowel syndrome patients.
Methods
Electronic databases were used to search for the following words and/or expressions: “FODMAP Diet,” “Low FODMAP diet,” “irritable bowel syndrome,” and “Diet in IBS.” The study was carried out between February and September 2017.
Results
The low-FODMAP diet consists of two phases: in the first phase, carbohydrates associated with symptom induction in IBS patients and with the evaluation of the improvement conditions are eliminated or reduced; in the second phase, the eliminated groups are gradually reintroduced according to the presented symptomatology.
Conclusions
The low-FODMAP diet restrains the intake of certain food, and it leads to significant improvement in the symptoms of irritable bowel syndrome patients. However, some nutritional deficiencies may occur, if there is inadequate nutritional guidance, highlighting the need for adequate dietary management
Blocking soluble guanylate cyclase could be the present and future of NO/cGMP inhibition for vasoplegia treatment
No full textHPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner
No full textAbstract\ud
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Background\ud
Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role.\ud
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Methods\ud
PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures.\ud
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Results\ud
HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin.\ud
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Conclusion\ud
This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.This work was supported by grants from: Ludwig Institute for Cancer Research; Fundação de Amparo a pesquisa do Estado de São Paulo (FAPESP) to Tatiana Rabachini (2003/14008–9), Enrique Boccardo (2010/20002–0), Katia Regina Perez (05/59142–2), Iolanda Midea Cuccovia, Luisa Lina Villa (2008/03232–1 and 2008/57889–1); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to Rubiana Andrade and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) to Rubiana Andrade and Luisa Lina Villa (573799/2008–3), Enrique Boccardo (480552–2011) and Iolanda Midea Cuccovia. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication
Internal migration of physicians who graduated in Brazil between 1980 and 2014
No full textAbstract\ud
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Background\ud
The internal migration of physicians from one place to another in the same country can unbalance the supply and distribution of these professionals in national health systems. In addition to economic, social and demographic issues, there are individual and professional factors associated with a physician’s decision to migrate. In Brazil, there is an ongoing debate as to whether opening medicine programmes in the interior of the country can induce physicians to stay in these locations. This article examines the migration of physicians in Brazil based on the location of the medical schools from which they graduated.\ud
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Methods\ud
A cross-sectional design based on secondary data of 275,801 physicians registered in the Regional Councils of Medicine (Conselhos Regionais de Medicina—CRMs) who graduated between 1980 and 2014. The evaluated outcome was migration, which was defined as moving away from the state where they completed the medicine programme to another state where they currently work or live.\ud
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Results\ud
57.3% of the physicians in the study migrated. The probability of migration ratio was greater in small grouped municipalities and lower in state capitals. 93.4% of the physicians who trained in schools located in cities with less than 100,000 inhabitants migrated. Fewer women (54.2%) migrated than men (60.0%). More than half of the physicians who graduated between 1980 and 2014 are in federative units different from the unit in which they graduated. Individual factors, such as age, gender, time of graduation and specialty, vary between the physicians who did or did not migrate.\ud
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Conclusions\ud
The probability of migration ratio was greater in small municipalities of the Southeast region and strong in the states of Tocantins, Acre and Santa Catarina. New studies are recommended to deepen understanding of the factors related to the internal migration and non-migration of physicians to improve human resource for health policies.This study takes place with the financial support Fundação Faculdade de\ud
Medicina (FFM), conselho Regional de Medicina do Estado de São Paulo\ud
(Cremesp) and Conselho Federal de Medicina (CFM), agreement (#0075/\ud
2015). Alex Jones Flores Cassenote was given a PhD Student scholarship\ud
from the São Paulo Research Foundation – FAPESP (proc. 2013/18158–0)
Mutation analysis of SLC26A4 (Pendrin) gene in a Brazilian sample of hearing-impaired subjects
No full textAbstract\ud
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Background\ud
Mutations in the SLC26A4 gene are associated with Pendred syndrome and autosomal recessive non-syndromic deafness (DFNB4). Both disorders have similar audiologic characteristics: bilateral hearing loss, often severe or profound, which may be associated with abnormalities of the inner ear, such as dilatation of the vestibular aqueduct or Mondini dysplasia. But, in Pendred syndrome (OMIM #274600), with autosomal recessive inheritance, besides congenital sensorineural deafness, goiter or thyroid dysfunctions are frequently present. The aim of this study was to determine whether mutations in SLC26A4 are a frequent cause of hereditary deafness in Brazilian patients.\ud
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Methods\ud
Microsatellite haplotypes linked to SLC26A4 were investigated in 68 families presenting autosomal recessive non-syndromic deafness. In the probands of the 16 families presenting segregation consistent with linkage to SLC26A4, Sanger sequencing of the 20 coding exons was performed. In an additional sample of 15 individuals with suspected Pendred syndrome, because of the presence of hypothyroidism or cochleovestibular malformations, the SLC26A4 gene coding region was also sequenced.\ud
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Results\ud
In two of the 16 families with indication of linkage to SLC26A4, the probands were found to be compound heterozygotes for probably pathogenic different mutations: three novel (c.1003 T > G (p. F335 V), c.1553G > A (p.W518X), c.2235 + 2 T > C (IVS19 + 2 T > C), and one already described, c.84C > A (p.S28R). Two of the 15 individuals with suspected Pendred syndrome because of hypothyreoidism or cochleovestibular malformations were monoallelic for likely pathogenic mutations: a splice mutation (IVS7 + 2 T > C) and the previously described c.1246A > C (p.T416P). Pathogenic copy number variations were excluded in the monoallelic cases and in those with normal results after Sanger sequencing. Additional mutations in the SLC26A4 gene or other definite molecular cause for deafness were not identified in the monoallelic patients, after exome sequencing.\ud
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Conclusions\ud
Biallelic pathogenic mutations in SLC26A4 explained ~ 3% of cases selected because of autosomal recessive deafness. Monoallelic mutations were present in ~ 13% of isolated cases of deafness with cochleovestibular malformations or suspected Pendred syndrome. These data reinforce the importance of mutation screening of SLC26A4 in Brazilian subjects and highlight the elevated frequency of monoallelic patients.This work was supported by National Council for Scientific and Technological Development (CNPq) from Brazil (141998/2013-0, 202310/2015-9 to JMP and 309647/2015-0 to EAW)
Biochemical characterization of a phospholipase A2 homologue from the venom of the social wasp Polybia occidentalis
No full textAbstract
Background
Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis.
Methods
P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation.
Results
The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896.47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues.
Conclusion
This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues
CEMiTool: a Bioconductor package for performing comprehensive modular co-expression analyses
No full textAbstract\ud
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Background\ud
The analysis of modular gene co-expression networks is a well-established method commonly used for discovering the systems-level functionality of genes. In addition, these studies provide a basis for the discovery of clinically relevant molecular pathways underlying different diseases and conditions.\ud
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Results\ud
In this paper, we present a fast and easy-to-use Bioconductor package named CEMiTool that unifies the discovery and the analysis of co-expression modules. Using the same real datasets, we demonstrate that CEMiTool outperforms existing tools, and provides unique results in a user-friendly html report with high quality graphs. Among its features, our tool evaluates whether modules contain genes that are over-represented by specific pathways or that are altered in a specific sample group, as well as it integrates transcriptomic data with interactome information, identifying the potential hubs on each network. We successfully applied CEMiTool to over 1000 transcriptome datasets, and to a new RNA-seq dataset of patients infected with Leishmania, revealing novel insights of the disease’s physiopathology.\ud
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Conclusion\ud
The CEMiTool R package provides users with an easy-to-use method to automatically implement gene co-expression network analyses, obtain key information about the discovered gene modules using additional downstream analyses and retrieve publication-ready results via a high-quality interactive report.FAPESP grants No 2012/19278-6, 2013/08216-2, 2015/20897-0, 2017/05762-7, 2014/19323-7, 2014/24162-2, 2015/25825-8;\ud
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CNPq grants 443719/2014-4;\ud
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CAPES grants No. 1496888 and No. 1572872\ud
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FONDECYT-CONICYT grant 11161020\ud
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PAI-CONICYT grant PAI7917002
MLL2/KMT2D and MLL3/KMT2C expression correlates with disease progression and response to imatinib mesylate in chronic myeloid leukemia
No full textAbstract\ud
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Background\ud
Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR–ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR–ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C.\ud
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Methods\ud
Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression.\ud
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Results\ud
In patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway.\ud
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Conclusion\ud
Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.CNPq (National Council of Technological and Scientific Development), CAPES (Coordination for the Improvement of Higher Education Personnel), FAPDF (Federal District Research Foundation), and FAPESP