Monitoring cancer prognosis, diagnosis and treatment efficacy using metabolomics and lipidomics

Introduction: Cellular metabolism is altered during cancer initiation and progression, which allows cancer cells to increase anabolic synthesis, avoid apoptosis and adapt to low nutrient and oxygen availability. The metabolic nature of cancer enables patient cancer status to be monitored by metabolomics and lipidomics. Additionally, monitoring metabolic status of patients or biological models can be used to greater understand the action of anticancer therapeutics. Objectives: Discuss how metabolomics and lipidomics can be used to (i) identify metabolic biomarkers of cancer and (ii) understand the mechanism-of-action of anticancer therapies. Discuss considerations that can maximize the clinical value of metabolic cancer biomarkers including case–control, prognostic and longitudinal study designs. Methods: A literature search of the current relevant primary research was performed. Results: Metabolomics and lipidomics can identify metabolic signatures that associate with cancer diagnosis, prognosis and disease progression. Discriminatory metabolites were most commonly linked to lipid or energy metabolism. Case–control studies outnumbered prognostic and longitudinal approaches. Prognostic studies were able to correlate metabolic features with future cancer risk, whereas longitudinal studies were most effective for studying cancer progression. Metabolomics and lipidomics can help to understand the mechanism-of-action of anticancer therapeutics and mechanisms of drug resistance. Conclusion: Metabolomics and lipidomics can be used to identify biomarkers associated with cancer and to better understand anticancer therapies

Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method

Direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (DI nESI FT-ICR MS)offers high mass accuracy and resolution for analyzing complex metabolite mixtures. High dynamic range across a wide mass range, however, can only be achieved at the expense of mass accuracy, since the large numbers of ions entering the ICR detector induce adverse spacecharge effects. Here we report an optimized strategy for wide-scan DI nESI FT-ICR MS that increases dynamic range but maintains high mass accuracy. It comprises the collection if multiple adjacent selected ion monitoring (SIM) windows that are stitched together using novel algorithms. The final SIM-stitching method, derived from several optimization experiments, comprises 21 adjoining SIM windows each of width m/z 30 (from m/z 70 to 500; adjacent windows overlap by m/z 10) with an automated gain control (AGC) target of 1 105 charges. SIMstitching and wide-scan range (WSR; Thermo Electron)were compared using a defined standard to assess mass accuracy and a liver extract to assess peak count and dynamic range. SIM-stitching decreased the maximum mass error by 1.3- and 4.3-fold, and increased the peak count by 5.3- and 1.8-fold, versus WSR (AGC targets of 1 x 105 and 5 x 105, respectively). SIM-stitching achieved an rms mass error of 0.18 ppm and detected over 3000 peaks in liver extract. This novel approach increases metabolome coverage, has very high mass accuracy, and at 5.5 min/sample is conducive for high- throughput metabolomics

Advanced biofilm staining techniques for TEM and SEM in geomicrobiology: Implications for visualizing EPS architecture, mineral nucleation, and microfossil generation

Microbial biofilms and mats have long been studied for their role in mineral precipitation reactions in natural environments. Scanning electron microscopy (SEM) is often used to characterize biofilms and their associated precipitates, however, conventional SEM sample preparation methods do not typically preserve the structure of the extracellular polymeric substances (EPS), which account for a large portion of biofilm material and play crucial roles in biofilm function and mineral nucleation. In the present investigation, EPS preservation and visualization using transmission electron microscopy (TEM) was explored using three biofilm fixation and staining protocols. Although aspects of these protocols were developed for preserving complex eukaryotic tissue samples, the heterogeneous, three-dimensional nature of biofilms make them suitable candidates for these sample processing techniques. The results suggest that cryofixation provides the best preservation of cyanobacteria-dominated biofilm structures. A staining protocol including six different pre-embedding stains allowed for TEM visualization of the EPS matrix that encompasses biofilm cells and precipitates. Of the stains used, uranyl acetate appears to be important in avoiding biofilm deformation during sample processing. Using these staining protocols, cell-EPS-mineral relationships were observed, including the precipitation of hydromagnesite [Mg₅(CO₃)₄(OH)₂·4H₂O] on the EPS adjacent to the exterior of cyanobacteria filaments. Beachrock-associated biofilms were characterized using both TEM of ultrathin sections, as well as SEM of resin embedded osmium stained biofilms prepared as petrographic thin sections. Combining these two approaches enabled characterization of both the micrometer-scale cell-carbonate mineral contacts, as well as the larger scale microbial colony-mineral cement relationships. These results suggest that sample preparation techniques developed for rapid preservation of eukaryotic tissue samples can be used to preserve and characterize biofilm architecture. These findings have applications to understanding mineral nucleation in biofilms, and the preservation of biofilms as microfossils in the rock record

Carbon Sequestration in Biogenic Magnesite and Other Magnesium Carbonate Minerals

The stability and longevity of carbonate minerals make them an ideal sink for surplus atmospheric carbon dioxide. Biogenic magnesium carbonate mineral precipitation from the magnesium-rich tailings generated by many mining operations could offset net mining greenhouse gas emissions, while simultaneously giving value to mine waste products. In this investigation, cyanobacteria in a wetland bioreactor enabled the precipitation of magnesite (MgCO3), hydromagnesite [Mg5(CO3)4(OH)2·4H2O], and dypingite [Mg5(CO3)4(OH)2·5H2O] from a synthetic wastewater comparable in chemistry to what is produced by acid leaching of ultramafic mine tailings. These precipitates occurred as micrometer-scale mineral grains and microcrystalline carbonate coatings that entombed filamentous cyanobacteria. This provides the first laboratory demonstration of low temperature, biogenic magnesite precipitation for carbon sequestration purposes. These findings demonstrate the importance of extracellular polymeric substances in microbially enabled carbonate mineral nucleation. Fluid composition was monitored to determine carbon sequestration rates. The results demonstrate that up to 238 t of CO2 could be stored per hectare of wetland/year if this method of carbon dioxide sequestration was implemented at an ultramafic mine tailing storage facility. The abundance of tailings available for carbonation and the anticipated global implementation of carbon pricing make this method of mineral carbonation worth further investigation

An infrared study of modern and paleo-filamentous bacteria from Rio Tinto, Spain

The Rio Tinto River Basin in southwestern Spain is a natural acidic (pH ~2.3) drainage system that supports a diversity of acid tolerant bacteria and eukaryotes with iron and sulfur- oxidizing prokaryotes performing chemolithotrophy and supporting anaerobic respiration [1, 2]. River terrace deposits formed over the past 2 Myr have preserved remnants of this unique biosphere, particularly microbial filaments, which provide templates for iron sulphate and iron oxide precipitation [1, 2]. This process of permineralization causes organic material to become trapped within a mineral matrix and preserved over geological time. This study analysed cultured filamentous bacteria, modern biofilms and sediments, and river terrace deposits spanning 2.1 Myr to assess the preservation of organics in this extreme environment over time, and the ability to correlate them with a contemporary culture. Filamentous bacteria are preserved within optically translucent nanophase to crystalline jarosite and goethite within all samples. The cultures contained 1 μm diameter filaments, some partially encrusted with iron oxides with visible cell walls, and others completely free of iron oxides, that are morphologically comparable to those preserved in the Rio Tinto rock record. Organic compounds (e.g. aliphatic hydrocarbons, amides and carboxylic acids) were detected at various levels within the culture and river terraces using mid-IR spectroscopy. Rio Tinto is a natural laboratory allowing living cells to be studied and correlated to morphological and biomolecular fossils in the geological record. These deposits will provide predictive tools for biomarker studies that may be extended to analogous environments on ancient Earth or even Mars. [1] Fernández-Remolar et al. (2005) Earth Planet Sci Lett 240,149-167. [2] Fernández-Remolar & Knoll (2008) Icarus 194,72-85

Microbial Populations of Stony Meteorites: Substrate Controls on First Colonizers

Finding fresh, sterilized rocks provides ecologists with a clean slate to test ideas about first colonization and the evolution of soils de novo. Lava has been used previously in first colonizer studies due to the sterilizing heat required for its formation. However, fresh lava typically falls upon older volcanic successions of similar chemistry and modal mineral abundance. Given enough time, this results in the development of similar microbial communities in the newly erupted lava due to a lack of contrast between the new and old substrates. Meteorites, which are sterile when they fall to Earth, provide such contrast because their reduced and mafic chemistry commonly differs to the surfaces on which they land; thus allowing investigation of how community membership and structure respond to this new substrate over time. We conducted 16S rRNA gene analysis on meteorites and soil from the Nullarbor Plain, Australia. We found that the meteorites have low species richness and evenness compared to soil sampled from directly beneath each meteorite. Despite the meteorites being found kilometers apart, the community structure of each meteorite bore more similarity to those of other meteorites (of similar composition) than to the community structure of the soil on which it resided. Meteorites were dominated by sequences that affiliated with the Actinobacteria with the major Operational Taxonomic Unit (OTU) classified as Rubrobacter radiotolerans. Proteobacteria and Bacteroidetes were the next most abundant phyla. The soils were also dominated by Actinobacteria but to a lesser extent than the meteorites. We also found OTUs affiliated with iron/sulfur cycling organisms Geobacter spp. and Desulfovibrio spp. This is an important finding as meteorites contain abundant metal and sulfur for use as energy sources. These ecological findings demonstrate that the structure of the microbial community in these meteorites is controlled by the substrate, and will not reach homeostasis with the Nullarbor community, even after ca. 35,000 years. Our findings show that meteorites provide a unique, sterile substrate with which to test ideas relating to first-colonizers. Although meteorites are colonized by microorganisms, the microbial population is unlikely to match the community of the surrounding soil on which they fall

Proteomic responses to gold(III)-toxicity in the bacterium Cupriavidus metallidurans CH34

Accepted 11th October 2016The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(i/iii)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(iii)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(iii)-chloride, 50 μM Cu(ii)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(iii)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(iii) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration samples.Carla M. Zammit, Florian Weiland, Joël Brugger, Benjamin Wade, Lyron Juan Winderbaum, Dietrich H. Nies, Gordon Southam, Peter Hoffmann and Frank Reit

Association of a functional microsatellite within intron 1 of the BMP5 gene with susceptibility to osteoarthritis

<p>Abstract</p> <p>Background</p> <p>In a previous study carried out by our group, the genotyping of 36 microsatellite markers from within a narrow interval of chromosome 6p12.3-q13 generated evidence for linkage and for association to female hip osteoarthritis (OA), with the most compelling association found for a marker within intron 1 of the bone morphogenetic protein 5 gene (<it>BMP5</it>). In this study, we aimed to further categorize the association of variants within intron 1 of <it>BMP5 </it>with OA through an expanded genetic association study of the intron and subsequent functional analysis of associated polymorphisms.</p> <p>Methods</p> <p>We genotyped 18 common polymorphisms including 8 microsatellites and 9 single nucleotide polymorphisms (SNPs) and 1 insertion/deletion (INDEL) from within highly conserved regions between human and mouse within intron 1 of <it>BMP5</it>. These markers were then tested for association to OA by a two-stage approach in which the polymorphisms were initially genotyped in a case-control cohort comprising 361 individuals with associated polymorphisms (<it>P </it>≤ 0.05) then genotyped in a second case-control cohort comprising 1185 individuals.</p> <p>Results</p> <p>Two <it>BMP5 </it>intron 1 polymorphisms demonstrated association in the combined case-control cohort of 1546 individuals (765 cases and 781 controls): microsatellite D6S1276 (<it>P </it>= 0.018) and SNP rs921126 (<it>P </it>= 0.013). Functional analyses in osteoblastic, chondrocytic, and adipocytic cell lines indicated that allelic variants of D6S1276 have significant effects on the transcriptional activity of the <it>BMP5 </it>promoter <it>in vitro</it>.</p> <p>Conclusion</p> <p>Variability in gene expression of <it>BMP5 </it>may be an important contributor to OA genetic susceptibility.</p