MicroRNAs are small ~22 nucleotide RNA sequences that are guided to the 3’ untranslated region (UTR) of protein-coding target mRNA sequences. One particular microRNA, miR155, plays a remarkable role in the immune system, where it is essential for mounting appropriate immune responses. However, its dysregulation has been identified in multiple inflammatory disorders such as Multiple Sclerosis (MS), arthritis, psoriasis and colitis. More specifically, miR-155 has been found to be elevated in the serum and brain lesions of MS patients. Importantly, therapeutic inhibition of miR-155 can inhibit progression of the MS disease model. One of us has identified that macrophages are major contributor to miR-155 elevation in the MS disease model, whilst its inhibition specifically in macrophages can limit the disease. Here macrophages were isolated from the femur and tibia of wild-type (WT) mice and mice with a knock-out (KO) of the gene regulating miR-155 production, and were cultured in-vitro and stimulated with lipopolysaccharide (LPS) to simulate an immune response. Cells were then prepared for spectral analysis by FTIR imaging with a Perkin-Elmer Spotlight 400 imaging microscope. After pre-processing the dimensionality of spectra were reduced using principal components analysis, kernel-PCA and universal manifold application and projection (UMAP) and classified using a support vector machine algorithm, delivering a classification performance approaching F1~0.89. Spectral features differentiating WT and KO classes were observed across the fingerprint region with no single spectral marker being the sole source of differentiation of the downstream molecular events. This study exemplifies the challenge in spectral discrimination of the complexity of molecular events in ex-vivo models of immune dysregulation.
Multi-modal spectroscopic analysis of biological systems may offer an improved overall non-invasive biophotonic metric of the status of the system, further enhancing the diagnostic and prognostic capabilities of these technologies. In the present study macrophages were extracted from wild-type mice and mice with a knock-out of the gene regulating miR-155, which has been observed to occur in patients with various autoimmune disorders, including multiple sclerosis (MS). Macrophages were stimulated in-vitro to produce an immune response and were then screened spectroscopically with FTIR and Raman spectroscopy (at 532nm and 660nm). Low, medium and high level data fusion strategies for classification of response to stimulation and miRNA regulation were piloted, using downstream principal components analysis-support vector machine classifiers to test the impact of these strategies on classification performance. These techniques allowed the development of a combined highlevel data-fusion, classification pipeline with a high level of classification accuracy (F1<0.9), with reduced variability in performance. Our proposed spectroscopic assay-data fusion strategy may provide an adjunct to clinical screening and diagnosis of various autoimmune disorders whose aetiology is associated with genetic dysregulation.
Severe radiation toxicity can continue years after the completion of radiotherapy for prostate cancer patients. Currently, it is impossible to predict before treatment which patients will experience these long-term side effects. New approaches based on vibrational spectroscopy have advantages over lymphocyte and genomic assays in terms of minimal sample preparation, speed and cost. A high throughput method has been developed to measure Raman spectra from liquid plasma in a cover glass bottomed 96 well plate. However, the Raman spectra can show contributions from glass and water. The current study aims to optimise pre-processing steps to improve classification performance.
Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for
the subcellular imaging of single cells. Although the potential of the technique has frequently been
demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected.
Although often employed, chemical fixation of cells can cause modifications to the molecular composition
and therefore influence the observations made. However, the weak contribution of water to Raman spectra
offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to
record highly specific spectra from cells in their original state. Unfortunately, in common 2-D culture
models, the contribution of the substrates to the spectra recorded requires significant data pre-processing
causing difficulties in developing automated methods for the correction of the spectra. Moreover, the 2-D
in vitro cell model is not ideal and dissimilarities between different optical substrates within in vitro cell
cultures results in morphological and functional changes to the cells. The interaction between the cells and
their microenvironment is crucial to their behavior but also their response to different external agents such
as radiation or anticancer drugs. In order to create an experimental model closer to the real conditions
encountered by the cell in vivo, 3-D collagen gels have been evaluated as a substrate for the spectroscopic
study of live cells. It is demonstrated that neither the medium used for cell culture nor the collagen gels
themselves contribute to the spectra collected. Thus the background contributions are reduced to that of the
water. Spectral measurements can be made in full cell culture medium, allowing prolonged measurement
times. Optimizations made in the use of collagen gels for live cells analysis by Raman spectroscopy are
encouraging and studying live cells within a collagenous microenvironment seems perfectly accessible.
Raman spectroscopy, as an evaluation of the products of ionising radiation exposure in biological systems, has been utilised mainly in the evaluation of the impact of exposure in tissue, cellular constituents and live animals. It has also been recently demonstrated that Raman spectroscopy can demonstrate key spectroscopic changes in the live cell associated with significant apoptotic and necrotic chemical damage. The present preliminary work utilises Raman spectroscopy at 514.5 nm to evaluate the results of exposure to γ-rays in HaCaT cells from a Co-60 therapy source, in tandem with other biological assays. The results demonstrate that Raman spectral changes may be correlated with changes in the cell also identified in parallel biochemical assays.
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