Keith R. Bambery

Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue.

The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages

ABSTRACT The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae. Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae.

A three-dimensional multivariate image processing technique for the analysis of FTIR spectroscopic images of multiple tissue sections

BackgroundThree-dimensional (3D) multivariate Fourier Transform Infrared (FTIR) image maps of tissue sections are presented. A villoglandular adenocarcinoma from a cervical biopsy with a number of interesting anatomical features was used as a model system to demonstrate the efficacy of the technique.MethodsFour FTIR images recorded using a focal plane array detector of adjacent tissue sections were stitched together using a MATLAB® routine and placed in a single data matrix for multivariate analysis using Cytospec™. Unsupervised Hierarchical Cluster Analysis (UHCA) was performed simultaneously on all 4 sections and 4 clusters plotted. The four UHCA maps were then stacked together and interpolated with a box function using SCIRun software.ResultsThe resultant 3D-images can be rotated in three-dimensions, sliced and made semi-transparent to view the internal structure of the tissue block. A number of anatomical and histopathological features including connective tissue, red blood cells, inflammatory exudate and glandular cells could be identified in the cluster maps and correlated with Hematoxylin & Eosin stained sections. The mean extracted spectra from individual clusters provide macromolecular information on tissue components.Conclusion3D-multivariate imaging provides a new avenue to study the shape and penetration of important anatomical and histopathological features based on the underlying macromolecular chemistry and therefore has clear potential in biology and medicine.

Synchrotron Fourier transform infrared (FTIR) analysis of single living cells progressing through the cell cycle

The application of FTIR spectroscopy to disease diagnosis requires a thorough knowledge of the spectroscopy associated with the cell cycle to discern disease markers from normal cellular events. We have applied synchrotron FTIR spectroscopy to monitor cells at different phases of the cell cycle namely G1, S and G2 phases. By applying Principal component analysis (PCA) from three independent trials we show clustering on a 2-dimensional scores plots (PC1 versus PC2) from cell spectra only two hours apart within the cell cycle. The corresponding PCA Loadings Plots indicate the clustering is primarily based on changes to the overall concentration of nucleic acids, proteins and lipids. During the first ten hours post mitosis, cells are observed to increase in protein and decrease in both lipid and nucleic acid concentration. During the synthesis phase, (beginning 9-11 hours post-mitosis) the PCA Loadings Plots show the accumulation of lipids within the cell as well the duplication of the genome as evidenced by strong DNA contributions. In the 4-6 hours following the synthesis phase, the cells once again accumulate protein while the relative nucleic acid and lipid concentrations decrease. These results, in comparison to previous studies on dehydrated cells, show previously unresolvable biochemical information as well as highlighting the advantages of FTIR spectroscopy applied to single living cells.

Importance of Tissue Preparation Methods in FTIR Micro-Spectroscopical Analysis of Biological Tissues: ‘Traps for New Users’

Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

Discriminating the intraerythrocytic lifecycle stages of the malaria parasite using synchrotron FT-IR microspectroscopy and an artificial neural network.

Synchrotron Fourier transform infrared (FT-IR) spectra of fixed single erythrocytes infected with Plasmodium falciparum at different stages of the intraerythrocytic cycle are presented for the first time. Bands assigned to the hemozoin moiety at 1712, 1664, and 1209 cm(-1) are observed in FT-IR difference spectra between uninfected erythrocytes and infected trophozoites. These bands are also found to be important contributors in separating the trophozoite spectra from the uninfected cell spectra in principal components analysis. All stages of the intraerythrocytic lifecycle of the malarial parasite, including the ring and schizont stage, can be differentiated by visual inspection of the C-H stretching region (3100-2800 cm(-1)) and by using principal components analysis. Bands at 2922, 2852, and 1738 cm(-1) assigned to the nu(asym)(CH(2) acyl chain lipids), nu(sym)(CH(2) acyl chain lipids), and the ester carbonyl band, respectively, increase as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage. Training of an artificial neural network showed that excellent automated spectroscopic discrimination between P. falciparum-infected cells and the control cells is possible. FT-IR difference spectra indicate a change in the production of unsaturated fatty acids as the parasite matures. The ring stage spectrum shows bands associated with cis unsaturated fatty acids. The schizont stage spectrum displays no evidence of cis bands and suggests an increase in saturated fatty acids. These results demonstrate that different phases of the P. falciparum intraerthyrocytic life cycle are characterized by different lipid compositions giving rise to distinct spectral profiles in the C-H stretching region. This insight paves the way for an automated infrared-based technology capable of diagnosing malaria at all intraerythrocytic stages of the parasites life cycle.

Detection of an en masse and reversible B- to A-DNA conformational transition in prokaryotes in response to desiccation.

The role that DNA conformation plays in the biochemistry of cells has been the subject of intensive research since DNA polymorphism was discovered. B-DNA has long been considered the native form of DNA in cells although alternative conformations of DNA are thought to occur transiently and along short tracts. Here, we report the first direct observation of a fully reversible en masse conformational transition between B- and A-DNA within live bacterial cells using Fourier transform infrared (FTIR) spectroscopy. This biospectroscopic technique allows for non-invasive and reagent-free examination of the holistic biochemistry of samples. For this reason, we have been able to observe the previously unknown conformational transition in all four species of bacteria investigated. Detection of this transition is evidence of a previously unexplored biological significance for A-DNA and highlights the need for new research into the role that A-DNA plays as a cellular defence mechanism and in stabilizing the DNA conformation. Such studies are pivotal in understanding the role of A-DNA in the evolutionary pathway of nucleic acids. Furthermore, this discovery demonstrates the exquisite capabilities of FTIR spectroscopy and opens the door for further investigations of cell biochemistry with this under-used technique.

Correlation of atomic force microscopy and Raman micro-spectroscopy to study the effects of ex vivo treatment procedures on human red blood cells

The effects of fixation and dehydration on the distribution of heme-based molecules inside red blood cells and the structural integrity of the cells have been investigated using Raman mapping and AFM topographic imaging. A strong correlation was observed between the thickness of the cells as determined from AFM images and the intensity of the characteristic heme bands in the Raman maps, demonstrating that heme compounds are relatively evenly distributed inside dried and fixed cells in the majority of cases. The exception occurred when cells were dried in phosphate buffered saline, where more hemichrome appears close to the periphery of the cell despite the AFM image showing a plateau like topography. Using neat formaldehyde solution as a fixative is inadequate for a complete structural preservation and results in diffusion of hemoglobin into the surrounding area. However, a mixture of formaldehyde (3%) and glutaraldehyde (0.1%) in buffer was found to be sufficient to retain the structural integrity of cells with minimal autofluorescence. This protocol was also suitable for red blood cells infected with Plasmodium falciparum parasites, and preserved the characteristic knob-like structures on the infected red blood cell surface.

Fourier transform infrared imaging and small angle x-ray scattering as a combined biomolecular approach to diagnosis of breast cancer

Fourier transform infrared (FTIR) microspectroscopic imaging and small angle x-ray scattering (SAXS) were combined to investigate the supramolecular structure of collagen from 27 tissue sections from patients undergoing mastectomy, excisional biopsy, or mammoplasty. Both techniques were correlated by matching the scattering profile from the SAXS data with the integrated area of the infrared collagen region (1300-1180 cm(-1)). The FTIR spectral profiles and multivariate analysis of various tissue components showed consistent differences between all major tissue components, particularly between cancer and normal tissue cells. Analysis of the SAXS data revealed broad differences between cancer and normal tissue, but were inconclusive due to the small sample size. Parameters were extracted from each technique in relation to their characterization of collagen to reveal a good correlation between the two techniques, which diagnostically parallels with gold-standard Hematoxylin and Eosin (H&E) stained sections. The results show that the integrated area of collagen region in the FTIR spectrum for cancerous samples is greater than that for noncancerous samples indicating collagen disorder. This supports the notion that collagen is structurally disrupted in cancer tissue consistent with the interpretation of the SAXS data. Overall, both these techniques successfully distinguished cancer from normal breast tissue. Integration of these two techniques was able to better segregate cancer as well as provide a more complete understanding of the differences in collagen on all structural levels during breast cancer development.

Quantification of DNA in simple eukaryotic cells using Fourier transform infrared spectroscopy

A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact cells making elucidation of the DNA bands difficult. We use known composition DNA/hemoglobin standards to successfully estimate the DNA content in avian erythrocyte nuclei (44.2%) and intact erythrocytes (12.8%). Furthermore we demonstrate that the absorption of cellular DNA does follow the Beer-Lambert Law and highlights the role of conformation and hydration in FTIR spectroscopy of biological samples.