Molecular Typing of Lung Adenocarcinoma on Cytological Samples Using a Multigene Next Generation Sequencing Panel

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.     

Infection in Health Personnel with High and Low Levels of Exposure in a Hospital Setting during the H1N1 2009 Influenza A Pandemic

A novel H1N1 influenza A virus caused the first pandemic of the 21^st century in 2009. Hospitals had an increased demand of health consultations, that made it difficult to estimate the incidence of infection in hospital personnel due to asymptomatic presentations and the under notification of cases. To estimate and compare the rate of exposure of high versus low risk health personnel to 2009 pandemic H1N1 (H1N1pdm2009) influenza A virus in a University Hospital in Chile, we performed a comparative and prospective study. Serum samples were obtained from 117 individuals that worked in the emergency room (ER) and the operating room (OR) during the peak of the pandemic. Antibody titers were determined by the hemagglutination inhibition (HI) assay. Of the samples analyzed, 65% were workers at the ER and 35% at the OR. Of the total number of the subjects tested, 29.1% were seropositive. One out of 3 (36.8%) workers at the ER had positive HI titers, meanwhile only 1 out of 7 (14.6%) workers from the OR was seropositive to the virus. The possibility of being infected in the ER as compared to the OR was 3.4 times greater (OR 3.4; CI 95%, 1.27–9.1), and the individuals of the ER had almost twice as much antibody titers against H1N1pdm2009 than the personnel in the OR, suggesting the potential of more than one exposure to the virus. Of the 34 seropositive subjects, 12 (35.3%) did not develop influenza like illness, including 2 non-clinical personnel involved in direct contact with patients at the ER. Considering the estimated population attack rate in Chile of 13%, both groups presented a higher exposure and seropositive rate than the general population, with ER personnel showing greater risk of infection and a significantly higher level of antibodies. This data provide a strong rationale to design improved control measures aimed at all the hospital personnel, including those coming into contact with the patients prior to triage, to prevent the propagation and transmission of respiratory viruses, particularly during a pandemic outbreak.     

Evidence of post-pollination barriers among three colour morphs of the deceptive orchid Dactylorhiza sambucina (L.) Soó

Floral-colour polymorphism in rewardless orchids has been hypothesized to be maintained by means of naïve insects, which after visiting a flower without reward will tend to fly elsewhere, looking for a flower of a different colour. In this study, levels of male and female reproductive success were monitored in Southern Italy populations of the deceptive orchid Dactylorhiza sambucina, through field observations over 3 years. These populations were characterized by the presence of a rare pink morph which is sympatric with the more frequent yellow and red morphs. In addition, final plant fertility was evaluated through percentages of embryo-containing seeds produced in both natural conditions and hand-pollination experiments. Results showed that pollinator preferences were independent of the morph frequencies and thus do not promote the predicted negative frequency-dependent selection. Although yellow and pink morphs showed significantly higher male reproductive success (RS), we found comparable levels of female RS, which suggest that pollinator behaviour cannot be the main mechanism which maintains this polymorphism. Interestingly, we found different percentages of embryo-containing seeds in fruits set under natural conditions as well as in those obtained from experimental crosses. In particular, pink morph showed a very low intrinsic fertility. Moreover, fertility in intra- was higher than in inter-morph crosses. To our knowledge, this is the first study pointing out the occurrence in the orchid family of post-pollination reproductive barriers. Findings are discussed in light of present hypothesis on the evolution and maintenance of colour polymorphism in deceptive orchids and other angiosperms.     

New implications on genomic adaptation derived from the Helicobacter pylori genome comparison

Background

Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium.

Principal Findings

We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome.

Conclusion

Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style.     

Functional diversity of marine macrobenthic communities from sublittoral soft-sediment habitats off northern Chile

Benthic communities show changes in composition and structure across different environmental characteristics and habitats. However, incorporating species biological traits into the analysis can provide a better understanding of system functioning within habitats. We compare the functional diversity of macrobenthic communities from a contrasting shallow (15 m) and deep (50 m) sublittoral soft-sediment habitats in northern Chile, using biological traits analysis. Our aim was to highlight the biological characteristics responsible for differences between habitats and the implications for ecosystem functioning. Trait analysis showed that the deep habitat was restricted in providing functionally important biogenic structure and bioturbation and supports less diverse feeding-related energy pathways. The shallow habitat is characterized by more diverse energy pathways and a higher potential for matter exchange through bioturbation. We provide support to the predictions of transfer of energy from the benthos to upper trophic levels in the shallow, which is characterized mainly by normoxia and little organic matter content in the sediment. In the deep habitat, characterized by hypoxia and more organic matter, energy appears to be transferred to microbial components. We suggest that trait analysis should be added to the traditional approaches based on species diversity, because it provides indicators of ecosystem stress.     

Do wood-based panels made with agro-industrial residues provide environmentally benign alternatives? An LCA case study of sugarcane bagasse addition to particle board manufacturing

Purpose

Sugarcane bagasse is one of the main agro-industrial residues which can be used to produce wood-based panels. However, more investigations related to its environmental performance assessment are needed, focusing on questions such as: Does it provide environmental benefits? What are its main environmental impacts? Could it substitute wood as raw material? Accordingly, this paper presents a life cycle assessment (LCA) study of particle board manufactured with sugarcane bagasse residues.

Methods

The cradle-to-gate assessment of 1 m³ of particle board made with sugarcane bagasse (PSB) considered three main subsystems: bagasse generation, bagasse distribution, and PSB production. For the inventory of PSB, dataset from two previous LCA studies related to the conventional particle board production and the ethanol life cycle for the Brazilian context were used. The allocation criterion for the bagasse generation subsystem was 9.08 % (economic base). The potential environmental impact phase was assessed by applying the CML and USEtox methods. PSB was compared with the conventional particle board manufactured in Brazil by the categories of the CML and USETox, and including land use indicators. Finally, two scenarios were analyzed to evaluate the influence of the allocation criteria and the consumption of sugarcane bagasse.

Results and discussion

All hotspots identified by CML and USETox methods are mainly related to the PSB production subsystem (24–100 % of impacts) due to heavy fuel oil, electricity, and urea-formaldehyde resin supply chain. The bagasse generation subsystem was more relevant to the eutrophication category (75 % of impacts). The bagasse distribution subsystem was not relevant because the impacts on all categories were lower than 1 %. PSB can substitute the conventional particle board mainly because of its lower contribution to abiotic depletion and ecotoxicity. Regarding land use impacts, PSB showed lower values according to all indicators (38–40 % of all impacts), which is explained by the lower demand for land occupation in comparison to that of the traditional particle board.

Conclusions

PSB can replace the traditional particle board due to its better environmental performance. The analysis of the economic allocation criterion was relevant only for the EP category, being important to reduce diesel and N-based fertilizers use during sugarcane cultivation. Regarding the influence of the sugarcane bagasse consumption, it is suggested that the sugarcane bagasse be mixed up to 75 % during particle board manufacturing so that good quality properties and environmental performance of panels can be provided.     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.     

Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.     

Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.