Machine learning utilising spectral derivative data improves cellular health classification through hyperspectral infra-red spectroscopy

The objective differentiation of facets of cellular metabolism is important for several clinical applications, including accurate definition of tumour boundaries and targeted wound debridement. To this end, spectral biomarkers to differentiate live and necrotic/apoptotic cells have been defined using in vitro methods. The delineation of different cellular states using spectroscopic methods is difficult due to the complex nature of these biological processes. Sophisticated, objective classification methods will therefore be important for such differentiation. In this study, spectral data from healthy/traumatised cell samples using hyperspectral imaging between 2500-3500 nm were collected using a portable prototype device. Machine learning algorithms, in the form of clustering, have been performed on a variety of pre-processing data types including 'raw' unprocessed, smoothed resampling, background subtracted and spectral derivative. The resulting clusters were utilised as a diagnostic tool for the assessment of cellular health and quantified using both sensitivity and specificity to compare the different analysis methods. The raw data exhibited differences for one of the three different trauma types applied, although unable to accurately cluster all the traumatised samples due to signal contamination from the chemical insult. The background subtracted and smoothed data sets reduced the accuracy further, due to the apparent removal of key spectral features which exhibit cellular health. However, the spectral derivative data-types significantly improved the accuracy of clustering compared to other data types, with both sensitivity and specificity for the background subtracted data set being >94% highlighting its utility to account for unknown signal contamination while maintaining important cellular spectral features

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence

Compressive sensing based spatial frequency domain imaging reconstruction

Compressive sensing methods demonstrate the ability to collect large data sets using a reduced number of observations. Here we present advantages of compressive sensing applied to Spatial Frequency Domain Imaging.</p

Cell trauma detection using infra-red live cell imaging

Infra-red (IR) spectroscopic imaging of live cells is greatly affected by the presence of water, which is a strong absorber of IR radiation. In order to overcome this, a variety of methods have been developed using complex microfluidic devices to reduce the liquid sample path length. However, these devices are often custom made needing both specialised equipment and detailed fabrication steps. Here we show the novel utilisation of a liquid-air interface configuration and a negative contrast imaging device (NCI) reflectance imaging system for the collection of spectral data from live cells within an in vitro environment. Spectral differences were observed between two different cell densities, both in the presence and absence of cell culture media. Additionally, differences were observed between control and test cultures exposed to dimethyl sulfoxide (DMSO) to induce cell apoptosis. The NCI system acquired data in the 2.5 – 3.5 µm spectral region, at a spectral sampling interval of 10 nm. This method will allow further investigation of spectral bio-markers within cell cultures to augment understanding of specific cell contributions to wound healing in vivo.</p

Mid-infrared spectroscopic imaging to assess wounded tissue health

Modern traumatic injuries, as encountered in battlefield conflicts, are often characterised by extensive soft tissue damage from blasts and high energy projectiles. This situation has created a challenge for wound stabilisation and repair, with surgical intervention common, via wound debridement procedures. These are often complex surgeries where necrotic and infected tissue is removed, usually with multiple remedial surgeries, designed to aid the natural healing process and to reduce the likelihood of infection. With extensive injuries, the preservation of viable tissue is paramount to functional recovery. Additionally, identifying wounds which are likely to heal without intervention, as well as those that exhibit precursors for impaired healing or infection, would assist in informing the appropriate medical care. Technologies that utilise concepts of non-contact imaging, such as optical imaging and spectroscopy can be used to obtain spatial and spectral maps of biomarkers, which provide valuable information on the wound (e.g. precursors to improper healing or delineate viable and necrotic tissue). A negative contrast imaging device (NCI) has been shown to characterise wound biopsies, through mid-IR (2.6 - 4 μm) non-invasive spectroscopic imaging. To better demonstrate the applicability of this technique, wound relevant cell cultures, subjected to induced trauma, are used to identify spectral changes between healthy and traumatised cells. This work highlights the available contrast in spectroscopic mid-IR signals and demonstrates the utility of spatially and spectrally derived maps as an assessment tool for wound diagnostics.</p

Blast wave exposure to the extremities causes endothelial activation and damage

Extremity injury is a significant burden to those injured in explosive incidents and local ischaemia can result in poor functionality in salvaged limbs. This study examined whether blast injury to a limb resulted in a change in endothelial phenotype leading to changes to the surrounding tissue. The hind limbs of terminally anaesthetized rabbitswere subjected to one of four blast exposures (high, medium, low, or no blast). Blood samples were analyzed for circulating endothelial cells pre-injury and at 1, 6, and 11 h postinjury as well as analysis for endothelial activation pre-injury and at 1, 6, and 12 h postinjury. Post-mortem tissue (12 h post-injury) was analysed for both protein and mRNA expression and also for histopathology. The high blast group had significantly elevated levels of circulating endothelial cells 6 h postinjury. This group also had significantly elevated tissue mRNA expression of IL-6, E-selectin, TNF-a, HIF-1, thrombomodulin, and PDGF. There was a significant correlation between blast dose and the degree of tissue pathology (hemorrhage, neutrophil infiltrate, and oedema) with the worst scores in the high blast group. This study has demonstrated that blast injury can activate the endothelium and in some cases cause damage that in turn leads to pathological changes in the surrounding tissue. For the casualty injured by an explosion the damaging effects of hemorrhage and shock could be exacerbated by blast injury and vice versa so that even low levels of blast become damaging, all of which could affect tissue functionality and long-term outcomes

BaTdp co-localises with cellular LC3.

<p>HEK293T cells were transfected with plasmid encoding GFP-tagged BaTdp. 24 hours post transfection, cells were fixed, permeabilised and stained with an anti-LC3B antibody and a secondary red fluorescent antibody, followed by analysis by fluorescence microscopy. Non-transfected cells, or cells transfected plasmid encoding GFP were used as control. All scale bars, 10 μm. Original magnification ×100.</p

Sequence alignment of TIR domain proteins.

<p>Sequence alignment of the TIR domains from BaTdp (BA_4098, residues 126–266), BwTdp from <i>Bacillus weihenstephanensis</i> (KEZ82977.1, residues 132–256), TcpB from <i>Brucella melitensis</i> (NP5404591.1, residues 116–250), TcpC from <i>Escherichia coli</i> CFT073 (NP_754290.1, residues 169–280), TcpF from <i>Enterococcus faecalis</i> (CCO72761.1, residues 3–160), TirS from <i>Staphylococcus aureus</i> (WP_000114516.1, residues 142–246), SaTlp1 from <i>S</i>. <i>aureus</i> (CAQ50581.1, residues 202–308), YpTdp from <i>Yersinia pestis</i> (NP_ 669733.1, residues 139–240), and human proteins MyD88 (AAH13589.1, residues 146–296), TLR2 (AAH33756.1, residues 641–784) and TLR4 (NP_003257.1, residues 621–781). The Box 1 and Box 2 regions are indicated by red boxes. The conserved glycine residue in the box 2 region is marked with an asterisk. Structural features indicated are based on the TcpB structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref019" target="_blank">19</a>]. The alignment was generated using MAFFT v.7220 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref035" target="_blank">35</a>] and visualised in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref036" target="_blank">36</a>].</p

Expression of BaTdp increases lipidation of cellular LC3.

<p>(A) HEK293T cells were transfected with plasmid encoding either BaTdp (wild type) or BaTdp (G164A) protein. After 24 or 48 hours post-transfection, cells were lysed in PBS containing 1% SDS and lysates were resolved by SDS-PAGE and analysed by Western blot using an anti-LC3B primary antibody or anti-GAPDH antibody for loading control. Result shown is representative of three independent experiments. Figures show cropped lanes from non-adjacent lanes of the same blot. Images have been cropped to only show the regions of interest. (Uncropped versions of blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.s001" target="_blank">S1 Fig</a>) (B) Western blots were analysed by densitometry using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref043" target="_blank">43</a>]. For each sample, the LC3B-II value was normalised against corresponding GAPDH value. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. *P < 0.05 (by two-tailed Student’s t-test).</p