High-level of viral genomic diversity in cervical cancers: a Brazilian study on human papillomavirus type 16

Invasive cervical cancer (ICC) is the third most frequent cancer among women worldwide and is associated with persistent infection by carcinogenic human papillomaviruses (HPVs). The combination of large populations of viral progeny and decades of sustained infection may allow for the generation of intra-patient diversity, in spite of the assumedly low mutation rates of PVs. While the natural history of chronic HPVs infections has been comprehensively described, within-host viral diversity remains largely unexplored. In this study we have applied next generation sequencing to the analysis of intra-host genetic diversity in ten ICC and one condyloma cases associated to single HPV16 infection. We retrieved from all cases near full-length genomic sequences. All samples analyzed contained polymorphic sites, ranging from 3 to 125 polymorphic positions per genome, and the median probability of a viral genome picked at random to be identical to the consensus sequence in the lesion was only 40%. We have also identified two independent putative duplication events in two samples, spanning the L2 and the L1 gene, respectively. Finally, we have identified with good support a chimera of human and viral DNA. We propose that viral diversity generated during HPVs chronic infection may be fueled by innate and adaptive immune pressures. Further research will be needed to understand the dynamics of viral DNA variability, differentially in benign and malignant lesions, as well as in tissues with differential intensity of immune surveillance. Finally, the impact of intralesion viral diversity on the long-term oncogenic potential may deserve closer attention.

Current Challenges in Modeling Cellular Metabolism

Mathematical and computational models play an essential role in understanding the cellular metabolism. They are used as platforms to integrate current knowledge on a biological system and to systematically test and predict the effect of manipulations to such systems. The recent advances in genome sequencing techniques have facilitated the reconstruction of genome-scale metabolic networks for a wide variety of organisms from microbes to human cells. These models have been successfully used in multiple biotechnological applications. Despite these advancements, modeling cellular metabolism still presents many challenges. The aim of this Research Topic is not only to expose and consolidate the state-of-the-art in metabolic modeling approaches, but also to push this frontier beyond the current edge through the introduction of innovative solutions. The articles presented in this e-book address some of the main challenges in the field, including the integration of different modeling formalisms, the integration of heterogeneous data sources into metabolic models, explicit representation of other biological processes during phenotype simulation, and standardization efforts in the representation of metabolic models and simulation results.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

KIAA1549: BRAF gene fusion and FGFR1 hotspot mutations are prognostic factors in pilocytic astrocytomas

Up to 20% of patients with pilocytic astrocytoma (PA) experience a poor outcome. BRAF alterations and Fibroblast growth factor receptor 1 (FGFR1) point mutations are key molecular alterations in Pas, but their clinical implications are not established. We aimed to determine the frequency and prognostic role of these alterations in a cohort of 69 patients with PAs. We assessed KIAA1549:BRAF fusion by fluorescence in situ hybridization and BRAF (exon 15) mutations by capillary sequencing. In addition, FGFR1 expression was analyzed using immunohistochemistry, and this was compared with gene amplification and hotspot mutations (exons 12 and 14) assessed by fluorescence in situ hybridization and capillary sequencing. KIAA1549:BRAF fusion was identified in almost 60% of cases. Two tumors harbored mutated BRAF. Despite high FGFR1 expression overall, no cases had FGFR1 amplifications. Three cases harbored a FGFR1 p.K656E point mutation. No correlation was observed between BRAF and FGFR1 alterations. The cases were predominantly pediatric (87%), and no statistical differences were observed in molecular alterations-related patient ages. In summary, we confirmed the high frequency of KIAA1549:BRAF fusion in PAs and its association with a better outcome. Oncogenic mutations of FGFR1, although rare, occurred in a subset of patients with worse outcome. These molecular alterations may constitute alternative targets for novel clinical approaches, when radical surgical resection is unachievable.

Who is who in anaerobic oil biodegradation?

[Excerpt] Anaerobic bioremediation is an important alternative for the common aerobic cleanup of subsurface petroleum-contaminated soil and water. Microbial communities involved in anaerobic oil biodegradation are scarcely studied, and only few mechanisms of anaerobic hydrocarbons degradation are described. In this work, microbial degradation of aliphatic hydrocarbons (AHC) was studied by using culture-dependent and culture-independent approaches. Hexadecane and hexadecene-degrading microbial communities were enriched under sulfate-reducing and methanogenic conditions. The microorganisms present in the enriched cultures were identified by 16S rRNA gene sequencing. (...)

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

A critical evaluation of methods for the reconstruction of tissue-specific models

Under the framework of constraint based modeling, genome-scale metabolic models (GSMMs) have been used for several tasks, such as metabolic engineering and phenotype prediction. More recently, their application in health related research has spanned drug discovery, biomarker identification and host-pathogen interactions, targeting diseases such as cancer, Alzheimer, obesity or diabetes. In the last years, the development of novel techniques for genome sequencing and other high-throughput methods, together with advances in Bioinformatics, allowed the reconstruction of GSMMs for human cells. Considering the diversity of cell types and tissues present in the human body, it is imperative to develop tissue-specific metabolic models. Methods to automatically generate these models, based on generic human metabolic models and a plethora of omics data, have been proposed. However, their results have not yet been adequately and critically evaluated and compared. This work presents a survey of the most important tissue or cell type specific metabolic model reconstruction methods, which use literature, transcriptomics, proteomics and metabolomics data, together with a global template model. As a case study, we analyzed the consistency between several omics data sources and reconstructed distinct metabolic models of hepatocytes using different methods and data sources as inputs. The results show that omics data sources have a poor overlapping and, in some cases, are even contradictory. Additionally, the hepatocyte metabolic models generated are in many cases not able to perform metabolic functions known to be present in the liver tissue. We conclude that reliable methods for a priori omics data integration are required to support the reconstruction of complex models of human cells.

Priming of a DNA metabarcoding approach for species identification and inventory in marine macrobenthic communities

Dissertação de mestrado em Molecular Genetics

Evaluation of inflammatory response induced by different types of daily contact lenses

Dissertação de mestrado em Genética Molecular

Quantifying the legacy of the Chinese Neolithic on the maternal genetic heritage of Taiwan and Island Southeast Asia

There has been a long-standing debate concerning the extent to which the spread of Neolithic ceramics and Malay-Polynesian languages in Island Southeast Asia (ISEA) were coupled to an agriculturally driven demic dispersal out of Taiwan 4000 years ago (4 ka). We previously addressed this question using founder analysis of mitochondrial DNA (mtDNA) control-region sequences to identify major lineage clusters most likely to have dispersed from Taiwan into ISEA, proposing that the dispersal had a relatively minor impact on the extant genetic structure of ISEA, and that the role of agriculture in the expansion of the Austronesian languages was therefore likely to have been correspondingly minor. Here we test these conclusions by sequencing whole mtDNAs from across Taiwan and ISEA, using their higher chronological precision to resolve the overall proportion that participated in the "out-of-Taiwan" mid-Holocene dispersal as opposed to earlier, postglacial expansions in the Early Holocene. We show that, in total, about 20 % of mtDNA lineages in the modern ISEA pool result from the "out-of-Taiwan" dispersal, with most of the remainder signifying earlier processes, mainly due to sea-level rises after the Last Glacial Maximum. Notably, we show that every one of these founder clusters previously entered Taiwan from China, 6-7 ka, where rice-farming originated, and remained distinct from the indigenous Taiwanese population until after the subsequent dispersal into ISEA.