Glycosylation spectral signatures for glioma grade discrimination using Raman spectroscopy.

BACKGROUND: Gliomas are the most common brain tumours with the high-grade glioblastoma representing the most aggressive and lethal form. Currently, there is a lack of specific glioma biomarkers that would aid tumour subtyping and minimally invasive early diagnosis. Aberrant glycosylation is an important post-translational modification in cancer and is implicated in glioma progression. Raman spectroscopy (RS), a vibrational spectroscopic label-free technique, has already shown promise in cancer diagnostics. METHODS: RS was combined with machine learning to discriminate glioma grades. Raman spectral signatures of glycosylation patterns were used in serum samples and fixed tissue biopsy samples, as well as in single cells and spheroids. RESULTS: Glioma grades in fixed tissue patient samples and serum were discriminated with high accuracy. Discrimination between higher malignant glioma grades (III and IV) was achieved with high accuracy in tissue, serum, and cellular models using single cells and spheroids. Biomolecular changes were assigned to alterations in glycosylation corroborated by analysing glycan standards and other changes such as carotenoid antioxidant content. CONCLUSION: RS combined with machine learning could pave the way for more objective and less invasive grading of glioma patients, serving as a useful tool to facilitate glioma diagnosis and delineate biomolecular glioma progression changes.

Time-resolved observations of vibrationally excited NO X 2Π (v') formed from collisional quenching of NO A 2Σ+ (v = 0) by NO X 2Π: evidence for the participation of the NO a 4Π state.

Time-resolved observations have been made of the formation of vibrationally excited NO X 2Π (v') following collisional quenching of NO A 2Σ+ (v = 0) by NO X 2Π (v = 0). Two time scales are observed, namely a fast production rate consistent with direct formation from the quenching of the electronically excited NO A state, together with a slow component, the magnitude and rate of formation of which depend upon NO pressure. A reservoir state formed by quenching of NO A 2Σ+ (v = 0) is invoked to explain the observations, and the available evidence points to this state being the first electronically excited state of NO, a 4Π. The rate constant for quenching of the a 4Π state to levels v' = 11-16 by NO is measured as (8.80 ± 1.1) × 10-11 cm3 molecule-1 s-1 at 298 K where the error quoted is two standard deviations, and from measurements of the increased formation of high vibrational levels of NO(X) by the slow process we estimate a lower limit for the fraction of self-quenching collisions of NO A 2Σ+ (v = 0) which lead to NO a 4Π as 19%.

Sensitive detection of HO radicals produced in an atmospheric pressure plasma using Faraday rotation cavity ring-down spectroscopy.

Cavity ring-down spectroscopy (CRDS) is a well-established, highly sensitive absorption technique whose sensitivity and selectivity for trace radical sensing can be further enhanced by measuring the polarization rotation of the intracavity light by the paramagnetic samples in the presence of a magnetic field. In this paper, we highlight the use of this Faraday rotation cavity ring-down spectroscopy (FR-CRDS) for the detection of HO2 radicals. In particular, we use a cold atmospheric pressure plasma jet as a highly efficient source of HO2 radicals and show that FR-CRDS in the near-infrared spectral region (1506 nm) has the potential to be a useful tool for studying radical chemistry. By simultaneously measuring ring-down times of orthogonal linearly polarized light, measurements of Faraday effect-induced rotation angles (θ) and absorption coefficients (α) are retrieved from the same data set. The Faraday rotation measurement exhibits better long-term stability and enhanced sensitivity due to its differential nature, whereby highly correlated noise between the two channels and slow drifts cancel out. The bandwidth-normalized sensitivities are αmin=2.2×10-11 cm-1 Hz-1/2 and θmin=0.62 nrad Hz-1/2. The latter corresponds to a minimum detectable (circular) birefringence of Δnmin=5×10-16 Hz-1/2. Using the overlapping qQ3(N = 4-9) transitions of HO2, we estimate limits of detection of 3.1 × 108 cm-3 based on traditional (absorption) CRDS methods and 6.7 × 107 cm-3 using FR-CRDS detection, where each point of the spectrum was acquired during 2 s. In addition, Verdet constants for pertinent carrier (He, Ar) and bulk (N2, O2) gases were recorded in this spectral region for the first time. These show good agreement with recent measurements of air and values extrapolated from reported Verdet constants at shorter wavelengths, demonstrating the potential of FR-CRDS for measurements of very weak Faraday effects and providing a quantitative validation to the computed rotation angles.

Comparative Analysis of Hydrate Nucleation for Methane and Carbon Dioxide.

Research in the field of hydrate formation requires more focus upon its modelling to enable the researchers to predict and assess the hydrate formation and its characteristics. The main focus of the study was to analyze the deviations induced in various parameters related to hydrate nucleation caused by the choice of different measuring correlations or methods of their sub-components. To serve this purpose under a range of operational conditions, parameters of hydrate nucleation such as rates of nucleation and crystal growth, critical radius of the nucleus, and theoretical induction time for carbon dioxide and methane were considered in this study. From these measurements, we have quantitatively compared the ease of hydrate formation in CO2 and CH4 systems in terms of nucleation while analyzing how various correlations for intermediate parameters were affecting the final output. Values of these parameters were produced under the considered bracket of operational conditions and distributed among six cases using both general and guest-gas specific correlations for gas dissolution and fugacity and their combinations. The isotherms and isobars produced from some of the cases differed from each other considerably. The rate of nucleation in one case showed an exponential deviation with a value over 1 × 1028 at 5 MPa, while the rest showed values as multiples of 106. These deviations explain how sensitive hydrate formation is to processing variables and their respective correlations, highlighting the importance of understanding the applicability of semi-empirical correlations. An attempt was made to define the induction time from a theoretical perspective and derive a relevant equation from the existing models. This equation was validated and analyzed within these six cases from the experimental observations.

Cavity enhanced liquid-phase stopped-flow kinetics.

The first application of liquid-phase broadband cavity enhanced spectroscopy (BBCEAS) to the measurement of stopped-flow kinetics is reported. The stopped-flow technique is widely used for the study of the kinetics of fast liquid-phase reactions down to millisecond timescales. UV-visible absorption spectroscopy is commonly used as the detection method. Increased sensitivity can potentially allow reactions which are too fast to be measured, to be studied by slowing down the reaction rate through the use of lower concentration of reactants. A simple low cost BBCEAS experimental setup was coupled to a commercial stopped-flow instrument. Comparative standard absorption measurements were also made using a UV-visible double-beam spectrometer as the detector. Measurements were made on the reaction of potassium ferricyanide with sodium ascorbate under pseudo-first order conditions at pH 8 and pH 9.2 A cavity enhancement factor (CEF) of 78 at 434 nm was obtained whilst the minimum detectable change in the absorption coefficient αmin(t), was 1.35 × 10-5 cm-1 Hz-1/2. The kinetic data at pH 9.2 was too fast to be measured using conventional spectroscopy, whilst the BBCEAS measurements allowed 30 fold lower concentration of reactants to be used which slowed down the reaction rate enough to allow the rate constant to be determined. The BBCEAS results showed a 58 fold improvement in sensitivity over the conventional measurements and also compared favourably with the relatively few previous liquid-phase cavity enhanced kinetic studies which have been performed using significantly more complex and expensive experimental setups.

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS.

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10(-5) cm(-1) Hz(-1/2). This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests.

A comparison of visible wavelength reflectance hyperspectral imaging and Acid Black 1 for the detection and identification of blood stained fingerprints.

Bloodstains are often encountered at scenes of violent crime and have significant forensic value for criminal investigations. Blood is one of the most commonly encountered types of biological evidence and is the most commonly observed fingerprint contaminant. Presumptive tests are used to test blood stain and blood stained fingerprints are targeted with chemical enhancement methods, such as acid stains, including Acid Black 1, Acid Violet 17 or Acid Yellow 7. Although these techniques successfully visualise ridge detail, they are destructive, do not confirm the presence of blood and can have a negative impact on DNA sampling. A novel application of visible wavelength hyperspectral imaging (HSI) is used for the non-contact, non-destructive detection and identification of blood stained fingerprints on white tiles both before and after wet chemical enhancement using Acid Black 1. The identification was obtained in a non-contact and non-destructive manner, based on the unique visible absorption spectrum of haemoglobin between 400 and 500nm. Results from the exploration of the selectivity of the setup to detect blood against ten other non-blood protein contaminants are also presented. A direct comparison of the effectiveness of HSI with chemical enhancement using Acid Black 1 on white tiles is also shown.

The non-contact detection and identification of blood stained fingerprints using visible wavelength reflectance hyperspectral imaging: Part 1.

Blood is one of the most commonly encountered types of biological evidence found at scenes of violent crime and one of the most commonly observed fingerprint contaminants. Current visualisation methods rely on presumptive tests or chemical enhancement methods. Although these can successfully visualise ridge detail, they are destructive, do not confirm the presence of blood and can have a negative impact on DNA sampling. A novel application of visible wavelength reflectance hyperspectral imaging (HSI) has been used for the detection and positive identification of blood stained fingerprints in a non-contact and non-destructive manner on white ceramic tiles. The identification of blood was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. HSI has been used to successfully visualise ridge detail in blood stained fingerprints to the ninth depletion. Ridge detail was still detectable with diluted blood to 20-fold dilutions. Latent blood stains were detectable to 15,000-fold dilutions. Ridge detail was detectable for fingerprints up to 6 months old. HSI was also able to conclusively distinguish blood stained fingerprints from fingerprints in six paints and eleven other red/brown media with zero false positives.

The non-contact detection and identification of blood stained fingerprints using visible wavelength hyperspectral imaging: Part II effectiveness on a range of substrates.

Biological samples, such as blood, are regularly encountered at violent crime scenes and successful identification is critical for criminal investigations. Blood is one of the most commonly encountered fingerprint contaminants and current identification methods involve presumptive tests or wet chemical enhancement. These are destructive however; can affect subsequent DNA sampling; and do not confirm the presence of blood, meaning they are susceptible to false positives. A novel application of visible wavelength reflectance hyperspectral imaging (HSI) has been used for the non-contact, non-destructive detection and identification of blood stained fingerprints across a range of coloured substrates of varying porosities. The identification of blood was based on the Soret γ band absorption of haemoglobin between 400 nm and 500 nm. Ridge detail was successfully visualised to the third depletion across light coloured substrates and the stain detected to the tenth depletion on both porous and non-porous substrates. A higher resolution setup for blood stained fingerprints on black tiles, detected ridge detail to the third depletion and the stain to the tenth depletion, demonstrating considerable advancements from previous work. Diluted blood stains at 1500 and 1000 fold dilutions for wet and dry stains respectively were also detected on pig skin as a replica for human skin.

Fingerprint composition and aging: A literature review.

Fingerprints have a key role in criminal investigations and are the most commonly used form of evidence worldwide. Significant gaps remain however, in the understanding of fingerprint chemistry, including enhancement reaction mechanisms and the effect of environmental variables and time on composition. Determining the age of a fingerprint is also a relatively unexplored area. A successful method, with reliable and quantitative estimates, would have numerous advantages. Previous unreliable methods have predominantly focused on enhancement success based on physical and chemical changes. This review explores variations in composition due to donor characteristics and environmental variables, and identifies gaps for further research. We also present a qualitative and quantitative summary of the effect of time on composition. Kinetics are presented where known, with summary schematics for reaction mechanisms. Previous studies exploring methods for determining the age of a fingerprint are also discussed, including their advantages and disadvantages. Lastly we propose a potentially more accurate and reliable methodology for determining fingerprint age based on quantitative kinetic changes to the composition of a fingerprint over time.

Estimating temperature exposure of burnt bone - A methodological review.

Forensic anthropologists are frequently confronted with the need to interpret burnt bone. Regardless of the context, one of the key factors for the correct interpretation of the remains and a reconstruction of the incidents leading to incineration is the estimation of the maximum exposure temperature. The recent years have seen an influx in experimental research focusing on temperature estimation, spanning from colour assessment, mechanical strength measurements, histology and structural observations, biochemical changes and crystallinity studies, vastly advancing the understanding of heat induced changes in bone, thus facilitating a more accurate interpretation. This paper draws together and evaluates all currently available methodologies for temperature estimation.

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains.

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle-Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.

Uncovering potential diagnostic and pathophysiological roles of α-synuclein and DJ-1 in melanoma.

BACKGROUND: Melanoma, the most lethal skin cancer type, occurs more frequently in Parkinson's disease (PD), and PD is more frequent in melanoma patients, suggesting disease mechanisms overlap. α-synuclein, a protein that accumulates in PD brain, and the oncogene DJ-1, which is associated with PD autosomal recessive forms, are both elevated in melanoma cells. Whether this indicates melanoma progression or constitutes a protective response remains unclear. We hereby investigated the molecular mechanisms through which α-synuclein and DJ-1 interact, suggesting novel biomarkers and targets in melanoma. METHODS: The Cancer Genome Atlas (TCGA) expression profiles derived from UCSC Xena were used to obtain α-synuclein and DJ-1 expression and correlated with survival in skin cutaneous melanoma (SKCM). Immunohistochemistry determined the expression in metastatic melanoma lymph nodes. Protein-protein interactions (PPIs) and molecular docking assessed protein binding and affinity with chemotherapeutic drugs. Further validation was performed using in vitro cellular models and ELISA immunoassays. RESULTS: α-synuclein and DJ-1 were upregulated in primary and metastatic SKCM. Aggregated α-synuclein was selectively detected in metastatic melanoma lymph nodes. α-synuclein overexpression in SK-MEL-28 cells induced the expression of DJ-1, supporting PPI and a positive correlation in melanoma patients. Molecular docking revealed a stable protein complex, with differential binding to chemotherapy drugs such as temozolomide, dacarbazine, and doxorubicin. Parallel reduction of both proteins in temozolomide-treated SK-MEL-28 spheroids suggests drug binding may affect protein interaction and/or stability. CONCLUSION: α-synuclein, together with DJ-1, may play a role in melanoma progression and chemosensitivity, constituting novel targets for therapeutic intervention, and possible biomarkers for melanoma.

High sensitivity liquid phase measurements using broadband cavity enhanced absorption spectroscopy (BBCEAS) featuring a low cost webcam based prism spectrometer.

Cavity enhanced techniques enable high sensitivity absorption measurements in the liquid phase but are typically more complex, and much more expensive, to perform than conventional absorption methods. The latter attributes have so far prevented a wide spread use of these methods in the analytical sciences. In this study we demonstrate a novel BBCEAS instrument that is sensitive, yet simple and economical to set up and operate. We use a prism spectrometer with a low cost webcam as the detector in conjunction with an optical cavity consisting of two R = 0.99 dielectric mirrors and a white light LED source for illumination. High sensitivity liquid phase measurements were made on samples contained in 1 cm quartz cuvettes placed at normal incidence to the light beam in the optical cavity. The cavity enhancement factor (CEF) with water as the solvent was determined directly by phase shift cavity ring down spectroscopy (PS-CRDS) and also by calibration with Rhodamine 6G solutions. Both methods yielded closely matching CEF values of ~60. The minimum detectable change in absorption (αmin) was determined to be 6.5 × 10(-5) cm(-1) at 527 nm and was limited only by the 8 bit resolution of the particular webcam detector used, thus offering scope for further improvement. The instrument was used to make representative measurements on dye solutions and in the determination of nitrite concentrations in a variation of the widely used Griess Assay. Limits of detection (LOD) were ~850 pM for Rhodamine 6G and 3.7 nM for nitrite, respectively. The sensitivity of the instrument compares favourably with previous cavity based liquid phase studies whilst being achieved at a small fraction of the cost hitherto reported, thus opening the door to widespread use in the community. Further means of improving sensitivity are discussed in the paper.

Demonstration of a novel laser-driven light source for broadband spectroscopy between 170 nm and 2.1 µm.

Combining broadband light sources with optical cavities is a well established approach to sensitive monitoring of trace species in both gas and liquid phases. Here we investigate for the first time the potential of a novel source based on laser-driven xenon plasma technology for spectroscopic studies of gaseous species over the 170-2100 nm spectral range.

Discrimination of Sri Lankan black teas using fluorescence spectroscopy and linear discriminant analysis.

BACKGROUND: The quality of teas is currently graded using trained tea tasters, whose evaluation can sometimes be subjective. In this study the simple fluorescence-based technique of total luminescence spectroscopy (TLS) in conjunction with data classification using principal component analysis (PCA) was applied to discriminate between teas from 11 different Sri Lankan plantations. Solvent extraction of the tea samples was followed by TLS to record excitation-emission matrices in the excitation range 250-590 nm and emission range 300-700 nm. RESULTS: The application of PCA and linear discriminant analysis (LDA) allowed the successful classification of all 11 teas using only the first two principal components. LDA demonstrated how the technique was able to discriminate between all teas correctly with 100% classification. CONCLUSION: Further development of this work could lead to a simple device that could be used by tea manufacturers instead of or alongside trained tea tasters to grade teas.

The age estimation of blood stains up to 30 days old using visible wavelength hyperspectral image analysis and linear discriminant analysis.

A novel application of visible wavelength hyperspectral image analysis has been applied to determine the age of blood stains up to 30 days old. Reflectance spectra from selected locations within the hyperspectral image, obtained from a portable instrument, were subjected to spectral pre-processing. This was followed by the application of a linear discriminant classification model, making estimations possible with an average error of ±0.27days for the first 7 days and an overall average error of ±1.17days up to 30 days. This is also the first reported study of the determination of the age of fresh blood stains (less than one day old) with an error of ±0.09h. The studies have been made under controlled conditions and represent, at this stage, proof of concept results but also are the most accurate age estimation results for measurements between 0 and 30 days reported to date. The results are consistent with well-established kinetic processes suggesting that the pre-processing stages described are revealing spectroscopic changes which are reliably following the time dependent oxidation of HbO2. The potential for parameterisation of environmental factors to make the method generally applicable at crime scenes is discussed, along with the developments required to further improve classification and to make the instrument genuinely portable.

FTIR spectroscopy as a convenient tool for detection and identification of airborne Cr(VI) compounds arising from arc welding fumes.

Given the significant presence of the carcinogenic Cr(VI) in arc welding fumes from stainless steels, it is also important, in addition to estimating the Cr(VI) levels, to identify Cr(VI) compounds, as it throws light on the mechanistic pathways towards fume formation. FTIR data is presented in this paper for arc welding fumes collected from Manual Metal Arc Welding (MMA), Flux Cored Arc Welding (FCAW) and Solid Wire Welding (Metal Inert/ Active Gas Welding [MIG/ MAG]). For MMA and FCAW samples, clear spectra corresponding to Na, K, dichromates was observed at wave number of around 725-740 cm-1 and at 890-900 cm-1. Chromate species were also observed at around 850-855 cm-1, as was evidence of CrO3 (chromium trioxide) too (950-970 cm-1). The identification of these compounds was done by carefully identifying the Cr-O-Cr anti-symmetric vibrations, the symmetric stretching of the CrO4 tetrahedra, and the stretching vibrations of the planar CrO3 structure for the chromium trioxide. All the above compounds were volatile, and present as nanoparticles in welding fumes, thereby potentially causing significant harm to the welders. Additionally, crystalline phases (Fe-Mn spinels) were also observed through powder XRD, and the data was compared with ion chromatography estimates for Cr(VI) and found to be consistent.

The diagnostic and prognostic potential of the EGFR/MUC4/MMP9 axis in glioma patients.

Glioblastoma is the most aggressive form of brain cancer, presenting poor prognosis despite current advances in treatment. There is therefore an urgent need for novel biomarkers and therapeutic targets. Interactions between mucin 4 (MUC4) and the epidermal growth factor receptor (EGFR) are involved in carcinogenesis, and may lead to matrix metalloproteinase-9 (MMP9) overexpression, exacerbating cancer cell invasiveness. In this study, the role of MUC4, MMP9, and EGFR in the progression and clinical outcome of glioma patients was investigated. Immunohistochemistry (IHC) and immunofluorescence (IF) in fixed tissue samples of glioma patients were used to evaluate the expression and localization of EGFR, MMP9, and MUC4. Kaplan-Meier survival analysis was also performed to test the prognostic utility of the proteins for glioma patients. The protein levels were assessed with enzyme-linked immunosorbent assay (ELISA) in serum of glioma patients, to further investigate their potential as non-invasive serum biomarkers. We demonstrated that MUC4 and MMP9 are both significantly upregulated during glioma progression. Moreover, MUC4 is co-expressed with MMP9 and EGFR in the proliferative microvasculature of glioblastoma, suggesting a potential role for MUC4 in microvascular proliferation and angiogenesis. The combined high expression of MUC4/MMP9, and MUC4/MMP9/EGFR was associated with poor overall survival (OS). Finally, MMP9 mean protein level was significantly higher in the serum of glioblastoma compared with grade III glioma patients, whereas MUC4 mean protein level was minimally elevated in higher glioma grades (III and IV) compared with control. Our results suggest that MUC4, along with MMP9, might account for glioblastoma progression, representing potential therapeutic targets, and suggesting the 'MUC4/MMP9/EGFR axis' may play a vital role in glioblastoma diagnostics.

A comparison between visible wavelength hyperspectral imaging and digital photography for the detection and identification of bloodstained footwear marks.

One of the first challenges that crime scene examiners have is determining if a substance is blood before performing analysis. Conventional methods of detecting blood involve the use of chemicals and different wavelengths of light in tandem with digital photography. However, these methods are destructive or provide false positives. Visible wavelength hyperspectral imaging (HSI) is a noncontact blood detection method that has been proven to provide accurate and reliable results. A novel application of this technique has been used for the detection and positive identification of bloodstained footwear marks, of different dilutions ranging from undiluted to 1:50 with distilled water, and on a range of substrates, and colors. Comparisons between HSI and conventional digital photography were made using a grading scale and analyzed using Mann-Whitney U-tests. The HSI technique was able to detect a statistically significant greater amount of tread detail on white tiles, laminate, carpet, and blue tiles compared with the digital photography technique, which was only superior on black tiles. Critically, the HSI technique was also able to determine that the footwear marks were made in blood. These results show that HSI will be useful in forensic investigations, where it is known that the perpetrator has walked through the victim's blood and left a trail of footwear marks at the crime scene. Even if the perpetrator had time to clean up afterward resulting in diluted stains, HSI would still be able to detect bloodstained footwear marks with a greater amount of detail compared with digital photography.