Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.     

Production of a monoclonal antibody by ascites, hollow fiber system, and transgenic plants for vaccine production using CB.Hep-1 mAb as a study case

Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production.     

Towards Monitoring Biodiversity in Amazonian Forests: How Regular Samples Capture Meso-Scale Altitudinal Variation in 25 km2 Plots

Background

Ecological monitoring and sampling optima are context and location specific. Novel applications (e.g. biodiversity monitoring for environmental service payments) call for renewed efforts to establish reliable and robust monitoring in biodiversity rich areas. As there is little information on the distribution of biodiversity across the Amazon basin, we used altitude as a proxy for biological variables to test whether meso-scale variation can be adequately represented by different sample sizes in a standardized, regular-coverage sampling arrangement.

Methodology/Principal Findings

We used Shuttle-Radar-Topography-Mission digital elevation values to evaluate if the regular sampling arrangement in standard RAPELD (rapid assessments (“RAP”) over the long-term (LTER [“PELD” in Portuguese])) grids captured patters in meso-scale spatial variation. The adequacy of different sample sizes (n = 4 to 120) were examined within 32,325 km²/3,232,500 ha (1293×25 km² sample areas) distributed across the legal Brazilian Amazon. Kolmogorov-Smirnov-tests, correlation and root-mean-square-error were used to measure sample representativeness, similarity and accuracy respectively. Trends and thresholds of these responses in relation to sample size and standard-deviation were modeled using Generalized-Additive-Models and conditional-inference-trees respectively. We found that a regular arrangement of 30 samples captured the distribution of altitude values within these areas. Sample size was more important than sample standard deviation for representativeness and similarity. In contrast, accuracy was more strongly influenced by sample standard deviation. Additionally, analysis of spatially interpolated data showed that spatial patterns in altitude were also recovered within areas using a regular arrangement of 30 samples.

Conclusions/Significance

Our findings show that the logistically feasible sample used in the RAPELD system successfully recovers meso-scale altitudinal patterns. This suggests that the sample size and regular arrangement may also be generally appropriate for quantifying spatial patterns in biodiversity at similar scales across at least 90% (≈5 million km²) of the Brazilian Amazon.     

Nucleo-cytoplasmic transport as a therapeutic target of cancer

Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.     

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using ¹⁴C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of ¹⁴C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)''s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.     

Anthropogenic Landscape in Southeastern Amazonia: Contemporary Impacts of Low-Intensity Harvesting and Dispersal of Brazil Nuts by the Kayapó Indigenous People

Brazil nut, the Bertholletia excelsa seed, is one of the most important non-timber forest products in the Amazon Forest and the livelihoods of thousands of traditional Amazonian families depend on its commercialization. B. excelsa has been frequently cited as an indicator of anthropogenic forests and there is strong evidence that past human management has significantly contributed to its present distribution across the Amazon, suggesting that low levels of harvesting may play a positive role in B. excelsa recruitment. Here, we evaluate the effects of Brazil nut harvesting by the Kayapó Indigenous people of southeastern Amazonia on seedling recruitment in 20 B. excelsa groves subjected to different harvesting intensities, and investigated if management by harvesters influences patterns of B. excelsa distribution. The number of years of low-intensity Brazil nut harvesting by the Kayapó over the past two decades was positively related to B. excelsa seedling density in groves. One of the mechanisms behind the higher seedling density in harvested sites seems to be seed dispersal by harvesters along trails. The Kayapó also intentionally plant B. excelsa seeds and seedlings across their territories. Our results show not only that low-intensity Brazil nut harvesting by the Kayapó people does not reduce recruitment of seedlings, but that harvesting and/or associated activities conducted by traditional harvesters may benefit B. excelsa beyond grove borders. Our study supports the hypothesis that B. excelsa dispersal throughout the Amazon was, at least in part, influenced by indigenous groups, and strongly suggests that current human management contributes to the maintenance and formation of B. excelsa groves. We suggest that changes in Brazil nut management practices by traditional people to prevent harvesting impacts may be unnecessary and even counterproductive in many areas, and should be carefully evaluated before implementation.     

Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.     

Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.