Recent advances of vibrational spectroscopy and chemometrics for forensic biological analysis.

Biological materials found at a crime scene are crucially important evidence for forensic investigation because they provide contextual information about a crime and can be linked to the donor-individuals through combination with DNA analysis. Applications of vibrational spectroscopy to forensic biological analysis have been emerging because of its advantageous characteristics such as the non-destructivity, rapid measurement, and quantitative evaluation, compared to most current methods based on histological observation or biochemical techniques. This review presents an overview of recent developments in vibrational spectroscopy for forensic biological analysis. We also emphasize chemometric techniques, which can elicit reliable and advanced analytical outputs from highly complex spectral data from forensic biological materials. The analytical subjects addressed herein include body fluids, hair, soft tissue, bones, and bioagents. Promising applications for various analytical purposes in forensic biology are presented. Simultaneously, future avenues of study requiring further investigation are discussed.

Phenotype Profiling for Forensic Purposes: Determining Donor Sex Based on Fourier Transform Infrared Spectroscopy of Urine Traces.

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special importance to the modern criminal investigation as a source of individualizing DNA evidence. However, individual identification of a urine donor is generally difficult because of the small amount of DNA. Therefore, the development of an innovative method to provide phenotype information about the urine donor-including sex-is highly desirable. In this study, we developed a multivariate discriminant model for the ATR FT-IR spectra of dry urine to identify the donor sex. Rigorous selection of significant wavenumbers on the spectrum using a genetic algorithm enabled superb discrimination performance for the model and conclusively indicated a chemical origin for donor sex differences, which was supported by physiological knowledge. Although further investigations need to be conducted, this proof-of-concept study demonstrates the great potential of the developed methodology for phenotype profiling based on the analysis of urine traces.

Spectral Mining for Discriminating Blood Origins in the Presence of Substrate Interference via Attenuated Total Reflection Fourier Transform Infrared Spectroscopy: Postmortem or Antemortem Blood?

Often in criminal investigations, discrimination of types of body fluid evidence is crucially important to ascertain how a crime was committed. Compared to current methods using biochemical techniques, vibrational spectroscopic approaches can provide versatile applicability to identify various body fluid types without sample invasion. However, their applicability is limited to pure body fluid samples because important signals from body fluids incorporated in a substrate are affected strongly by interference from substrate signals. Herein, we describe a novel approach to recover body fluid signals that are embedded in strong substrate interferences using attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy and an innovative multivariate spectral processing. This technique supported detection of covert features of body fluid signals, and then identified origins of body fluid stains on substrates. We discriminated between ATR FT-IR spectra of postmortem blood (PB) and those of antemortem blood (AB) by creating a multivariate statistics model. From ATR FT-IR spectra of PB and AB stains on interfering substrates (polyester, cotton, and denim), blood-originated signals were extracted by a weighted linear regression approach we developed originally using principal components of both blood and substrate spectra. The blood-originated signals were finally classified by the discriminant model, demonstrating high discriminant accuracy. The present method can identify body fluid evidence independently of the substrate type, which is expected to promote the application of vibrational spectroscopic techniques in forensic body fluid analysis.

Advanced forensic validation for human spermatozoa identification using SPERM HY-LITER™ Express with quantitative image analysis.

Identification of human semen is indispensable for the investigation of sexual assaults. Fluorescence staining methods using commercial kits, such as the series of SPERM HY-LITER™ kits, have been useful to detect human sperm via strong fluorescence. These kits have been examined from various forensic aspects. However, because of a lack of evaluation methods, these studies did not provide objective, or quantitative, descriptions of the results nor clear criteria for the decisions reached. In addition, the variety of validations was considerably limited. In this study, we conducted more advanced validations of SPERM HY-LITER™ Express using our established image analysis method. Use of this method enabled objective and specific identification of fluorescent sperm's spots and quantitative comparisons of the sperm detection performance under complex experimental conditions. For body fluid mixtures, we examined interference with the fluorescence staining from other body fluid components. Effects of sample decomposition were simulated in high humidity and high temperature conditions. Semen with quite low sperm concentrations, such as azoospermia and oligospermia samples, represented the most challenging cases in application of the kit. Finally, the tolerance of the kit against various acidic and basic environments was analyzed. The validations herein provide useful information for the practical applications of the SPERM HY-LITER™ Express kit, which were previously unobtainable. Moreover, the versatility of our image analysis method toward various complex cases was demonstrated.

The applicability of ELISA detection of gastric mucosa-expressing proteins for the identification of vomit.

The identification of vomit stains may be helpful for crime scene reconstruction. However, there is no specific and convenient method for identifying vomit stain. Therefore, to establish the procedure for forensic identification of vomit stains, we focused on four gastric mucosa-expressing proteins, pepsinogen I (PGA), pepsinogen II (PGC), gastrin (GAST), and mucin 5AC (MUC5AC). We developed enzyme-linked immunosorbent assay (ELISA) procedures for the detection of these four candidate proteins. The specificity and sensitivity of ELISA detection of these proteins were analyzed, and applicability for the identification of vomit in forensic casework samples was also investigated. We found the sensitivities of ELISA for detection of PGA, PGC, GAST, and MUC5AC from the standard protein (peptide) and from diluted gastric mucosa extract were 10.0-100.0 ng/ml and 1:200-1:1600, respectively. PGA and PGC were successfully detected in stomach contents and gastric mucosa samples; however, these also cross-reacted with some urine and semen samples, respectively, because of low level expression in these fluids. MUC5AC was positive for most gastric mucosa samples; however, it was difficult to detect in stomach contents. ELISA detection of GAST was not suitable for the identification of vomit. All aged samples stored up to 90 days gave positive results for ELISA procedures for PGA, PGC, and MUC5AC. Therefore, ELISA detection of these proteins might be applicable to aged samples. PGA was also detected in all actual vomit samples tested. These results suggest that ELISA for the detection of gastric mucosa-expressing proteins, especially PGA, could be an effective tool for the forensic identification of vomit.

Practical evaluation of an RNA-based saliva identification method.

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein.

Studies of temporal behaviors of protein association in living cells are crucially important for elucidating the fundamental roles and the mechanism of interactive coordination for cell activities. We developed a method for investigating the temporal alternation of a particular protein assembly using monomeric fluorescent proteins, fluorescent timers (FTs), of which the fluorescent color changes from blue to red over time. We identified a dissection site of the FTs, which allows complementation of the split FT fragments. The split fragments of each FT variant recovered their fluorescence and maintained inherent rates of the color changes upon the reassembly of the fragments in vitro. We applied this method to visualize the aggregation process of α-synuclein in living cells. The size of the aggregates with the temporal information was analyzed from ratio values of the blue and red fluorescence of the reconstituted FTs, from which the aggregation rates were evaluated. This method using the split FT fragments enables tracing and visualizing temporal alternations of various protein associations by single fluorescence measurements at a given time point.

Large-scale omics dataset of polymer degradation provides robust interpretation for microbial niche and succession on different plastisphere.

While biodegradable polymers have received increased attention due to the recent marine plastic problem, few studies have compared microbiomes and their degradation processes among biodegradable polymers. In this study, we set up prompt evaluation systems for polymer degradation, allowing us to collect 418 microbiome and 125 metabolome samples to clarify the microbiome and metabolome differences according to degradation progress and polymer material (polycaprolactone [PCL], polybutylene succinate-co-adipate [PBSA], polybutylene succinate [PBS], polybutylene adipate-co-terephthalate [PBAT], and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [PHBH]). The microbial community compositions were converged to each polymer material, and the largest differences were observed between PHBH and other polymers. Such gaps were probably formed primarily by the presence of specific hydrolase genes (i.e., 3HB depolymerase, lipase, and cutinase) in the microorganisms. Time-series sampling suggested several steps for microbial succession: (1) initial microbes decrease abruptly after incubation starts; (2) microbes, including polymer degraders, increase soon after the start of incubation and show an intermediate peak; (3) microbes, including biofilm constructers, increase their abundance gradually. Metagenome prediction showed functional changes, where free-swimming microbes with flagella adhered stochastically onto the polymer, and certain microbes started to construct a biofilm. Our large-dataset-based results provide robust interpretations for biodegradable polymer degradation.

Integrative measurement analysis via machine learning descriptor selection for investigating physical properties of biopolymers in hairs.

Integrative measurement analysis of complex subjects, such as polymers is a major challenge to obtain comprehensive understanding of the properties. In this study, we describe analytical strategies to extract and selectively associate compositional information measured by multiple analytical techniques, aiming to reveal their relationships with physical properties of biopolymers derived from hair. Hair samples were analyzed by multiple techniques, including solid-state nuclear magnetic resonance (NMR), time-domain NMR, Fourier transform infrared spectroscopy, and thermogravimetric and differential thermal analysis. The measured data were processed by different processing techniques, such as spectral differentiation and deconvolution, and then converted into a variety of "measurement descriptors" with different compositional information. The descriptors were associated with the mechanical properties of hair by constructing prediction models using machine learning algorithms. Herein, the stepwise model refinement via selection of adopted descriptors based on importance evaluation identified the most contributive descriptors, which provided an integrative interpretation about the compositional factors, such as α-helix keratins in cortex; and bounded water and thermal resistant components in cuticle. These results demonstrated the efficacy of the present strategy to generate and select descriptors from manifold measured data for investigating the nature of sophisticated subjects, such as hair.

Soft and Robust Identification of Body Fluid Using Fourier Transform Infrared Spectroscopy and Chemometric Strategies for Forensic Analysis.

Body fluid (BF) identification is a critical part of a criminal investigation because of its ability to suggest how the crime was committed and to provide reliable origins of DNA. In contrast to current methods using serological and biochemical techniques, vibrational spectroscopic approaches provide alternative advantages for forensic BF identification, such as non-destructivity and versatility for various BF types and analytical interests. However, unexplored issues remain for its practical application to forensics; for example, a specific BF needs to be discriminated from all other suspicious materials as well as other BFs, and the method should be applicable even to aged BF samples. Herein, we describe an innovative modeling method for discriminating the ATR FT-IR spectra of various BFs, including peripheral blood, saliva, semen, urine and sweat, to meet the practical demands described above. Spectra from unexpected non-BF samples were efficiently excluded as outliers by adopting the Q-statistics technique. The robustness of the models against aged BFs was significantly improved by using the discrimination scheme of a dichotomous classification tree with hierarchical clustering. The present study advances the use of vibrational spectroscopy and a chemometric strategy for forensic BF identification.

Evaluation of skin- or sweat-characteristic mRNAs for inferring the human origin of touched contact traces.

The source of small amounts of touch DNA, which is transferred from the skin to an object when it is handled or touched, could be an issue in the forensic analysis of criminal cases. Here, we performed an extended evaluation of skin- or sweat-characteristic mRNAs to investigate their usability to infer whether an object has been handled or touched by someone. First, we compared the expression levels of candidate genes between skin swabs and other body fluids by quantitative RT-PCR analysis. Among the analyzed genes, corneodesmosin (CDSN), late cornified envelope 1C (LCE1C), filaggrin (FLG), desmocollin 1, and dermcidin were selected for further analysis on the basis of their specificities and sensitivities. Then, we tried to detect these genes from mock casework samples. As a result, CDSN, LCE1C, and FLG could be good markers because of their detectability. Finally, we determined the correlation between the expression of these genes and DNA yield of skin swabs to assess their adaptability as a screening test for touch DNA samples. However, the detectability of these genes was not correlated with the DNA yield of skin swab samples. In conclusion, gene expression analysis of the skin- or sweat-characteristic mRNAs CDSN, LCE1C, and FLG could be useful for inferring the skin origin of touched contact traces, but the use of the expression levels of these mRNAs for the prediction of DNA yield is problematic. To develop a screening test for touch DNA samples, other markers that have a well-correlated sensitivity with DNA analysis should be investigated.

Evaluation of a blood-specific DNA methylated region and trial for allele-specific blood identification from mixed body fluid DNA.

The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations.