Analysis of the fluorescent properties of vaginal fluid upon ageing.

Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman's menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.

Towards Onsite Age Estimation of Semen Stains Using Fluorescence Spectroscopy.

The age estimation of biological traces is one of the holy grails in forensic investigations. We developed a method for the age estimation of semen stains using fluorescence spectroscopy in conjunction with a stoichiometric ageing model. The model describes the degradation and generation rate of proteins and fluorescent oxidation products (FOX) over time. The previously used fluorimeter is a large benchtop device and requires system optimization for forensic applications. In situ applications have the advantage that measurements can be performed directly at the crime scene, without additional sampling or storage steps. Therefore, a portable fiber-based fluorimeter was developed, consisting of two optimized light-emitting diodes (LEDs) and two spectrometers to allow the fluorescence protein and FOX measurements. The handheld fiber can be used without touching the traces, avoiding the destruction or contamination of the trace. In this study, we have measured the ageing kinetics of semen stains over time using both our portable fluorimeter and a laboratory benchtop fluorimeter and compared their accuracies for the age estimation of semen stains. Successful age estimation was possible up to 11 days, with a mean absolute error of 1.0 days and 0.9 days for the portable and the benchtop fluorimeters, respectively. These results demonstrate the potential of using the portable fluorimeter for in situ applications.

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy.

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV-visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.

The compatibility of immunolabeling with STR profiling.

Immunolabeling is a technique, which has recently been introduced to enhance the quality of developed fingermarks and subsequently strengthen the evidential value. The effect of this method on subsequent DNA analysis, however, has not been explored yet. Therefore, the current pilot study aimed to determine whether STR profiling is possible after immunolabeling. Since immunolabeling involves washing steps which could reduce DNA quantities, the use of different fixatives including methanol, formaldehyde and universal molecular fixative (UMFIX) were investigated. STR profiles from the (immunolabeled) fingermarks were generated after four days and four weeks by a direct PCR method to enable comparison of relatively fresh and old fingermarks. The fingermarks were deposited on diverse forensically relevant substrates, including glass, metal and tile. STR profiles could be recovered for all tested fixatives with no significant difference in performance. However, the mean number of detected alleles was the highest when methanol was used for fixation. Furthermore, immunolabeling on aged fingermarks (4 weeks) was also possible, but the number of detected alleles showed a non-significant decrease. DNA could be recovered from deposits on all substrates, of which glass showed the highest mean number of detected alleles followed by metal and tile.

Estimating the Time of Deposition of Semen Traces using Fluorescence Protein-Lipid Oxidation Signatures.

In the forensic field, knowledge about the time of deposition of semen traces is extremely valuable to law enforcement agencies to assess the relevance of the traces and the validity of witness testimonies. However, currently, no method exists that is able to estimate the time of deposition of semen stains, due to the complex chemistry of the constituents and variation in degradation patterns. Here, we demonstrate a non-contact age estimation method to assess the time of deposition of semen stains using fluorescence spectroscopy. Protein-lipid oxidation reactions were monitored in semen stains over time using protein fluorescence and fluorescent oxidation product signatures to reveal distinctive aging patterns. On the basis of the relative amounts of these fluorescent products and the rate at which they are converted, successful age estimation was achieved up to a value of 16 days, with a median absolute error of 1.7 days. We demonstrate here a new tool that can fill the current gap in intelligence about the age of semen traces that has been challenging the forensic community worldwide.

Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid ß-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.