A comprehensive review on application of atomic force microscopy in Forensic science.

The primary objective of forensic investigation of a case is to recognize, identify, locate, and examine the evidence. Microscopy is a technique that provides crucial information for resolving a case or advancing the investigation process by analyzing the evidence obtained from a crime scene. It is often used in conjunction with suitable analytical techniques. Various microscopes are employed; scanning probe microscopes are available in diverse forensic analyses and studies. Among these, the atomic force microscope (AFM) is the most commonly used scanning probe technology, offering a unique morphological and physico-chemical perspective for analyzing multiple pieces of evidence in forensic investigations. Notably, it is a non-destructive technique capable of operating in liquid or air without complex sample preparation. The article delves into a detailed exploration of the applications of AFM in the realms of nanomechanical forensics and nanoscale characterization of forensically significant samples.

Signal Denoising of Traffic Speed Deflectometer Measurement Based on Partial Swarm Optimization-Variational Mode Decomposition Method.

To accurately identify the deflection data collected by a traffic speed deflectometer (TSD) and eliminate the noise in the measured signals, a TSD signal denoising method based on the partial swarm optimization-variational mode decomposition (PSO-VMD) method is proposed. Initially, the VMD algorithm is used for modal decomposition, calculating the correlation coefficients between each decomposed mode and the original signal for modal selection and signal reconstruction; Then, the particle swarm optimization algorithm is utilized to optimize the number of modes K and the value α for the VMD algorithm, adopting fuzzy entropy as the affinity function to circumvent effects from sequence decomposition and forecasting accuracy, thus identifying the optimal combination of hyperparameters. Finally, the analysis on simulated signals indicates that the PSO-VMD method secures the best parameters, showing a clear advantage in denoising. Denoising real TSD data validates that the approach proposed herein achieves commendable outcomes in TSD deflection noise reduction, offering a feasible strategy for TSD signal denoising.

Assessment of interproximal enamel reduction planned by the digital set-up of a customized lingual orthodontic appliance: A comparison cohort study.

Objectives: Interproximal enamel reduction (IER), commonly known as stripping, is a frequently used technique in orthodontic treatment to address issues related to arch length discrepancies and tooth size discrepancies (TSD). The use of digital set-up allows for precise prediction of the amount of IER required. TSD occurs when the sizes of maxillary and mandibular teeth are not in proportion to each other. This study aims to evaluate and compare the suggested IER values generated by the digital set-up of a customized lingual orthodontic appliance in both upper and lower arches, across sextants, and among different teeth concerning TSD. Materials and methods: We analyzed suggested IER values from 809 cases. The statistical analysis was divided into two parts: part 1 focused on the number of stripped surfaces, and part 2 assessed the quantity of enamel removed. Comparisons were made between upper and lower arches, sextants, and teeth using the Friedman test, followed by pairwise Wilcoxon tests with Bonferroni correction. Results: The study found that mandibular and frontal stripping were more frequently suggested than maxillary and posterior stripping. Lower canines were the teeth most commonly recommended for stripping, followed by upper incisors. Conclusion: Within the scope and limits of this cohort study, we conclude that, in general, more IER is required in the mandible as compared to the maxilla. Particularly in the anterior sextants, IER might be necessary to achieve optimal alignment and occlusion.

Response of salivary microbiome to temporal, environmental, and surface characteristics under in vitro exposure.

The microbiome of saliva stains deposited at crime scenes and in everyday settings is valuable for forensic investigations and environmental ecology. However, the dynamics and applications of microbial communities in these saliva stains have not been fully explored. In this study, we analyzed saliva samples that were exposed to indoor conditions for up to 1 year and to different carriers (cotton, sterile absorbent cotton swab, woolen, dacron) in both indoor and outdoor environments for 1 month using high-throughput sequencing. The analysis of microbial composition and Mfuzz clustering showed that the salivary flora, specifically Streptococcus (cluster7), which was associated with microbial contamination, remained stable over short periods of time. However, prolonged exposure led to significant differences due to the invasion of environmental bacteria such as Pseudomonas and Achromobacter. The growth and colonization of environmental flora were promoted by humidity. The neutral model predictions indicated that the assembly of salivary microbial communities in outdoor environments was significantly influenced by stochastic processes, with environmental characteristics having a greater impact on community change compared to surface characteristics. By incorporating data from previous studies on fecal and vaginal secretion microbiology, we developed RF and XGBoost classification models that achieved high accuracy (>98 %) and AUC (>0.8). Additionally, a RF regression model was created to determine the time since deposition (TsD) of the stains. Time inference models yielded a mean absolute error (MAE) of 7.1 days for stains exposed for 1 year and 14.2 h for stains exposed for 14 days. These findings enhance our understanding of the changes in the microbiome of saliva stains over time, in different environments, and on different surfaces. They also have potential applications in assessing potential microbial contamination, identifying body fluids, and inferring the time of deposition.

Transdiagnostic effects of therapist self-disclosure on diverse emotional experiences of clients with emotional disorders and schizophrenia.

OBJECTIVE: Despite the clinical significance of emotional diversity, also known as emodiversity, there has been limited investigation into the therapeutic interventions that influence this construct. In the current study we examined the association between immediate therapist self-disclosure (TSD) and emodiversity among two diagnostic groups who tend to experience emotional difficulties: people with schizophrenia and people with emotional disorders (i.e., depression and/or anxiety). METHOD: The sample comprised 74 clients (37 diagnosed with schizophrenia and 37 with emotional disorders) treated by 45 therapists in a university clinic setting. Following each session, clients self-reported their emotions, and therapists completed a measure of frequency and centrality of their immediate TSD during the session. RESULTS: Longitudinal multilevel models indicated that immediate TSD was positively associated with clients' global emodiversity, both at the within- and the between-client levels, as well as with clients' negative emodiversity at the between-client level. Moreover, clients with emotional disorders and clients with schizophrenia did not differ in the association between immediate TSD and emodiversity. In addition, across groups, clients treated by therapists who used more immediate TSD on average showed greater increases in global emodiversity during treatment. CONCLUSIONS: immediate TSD is associated with clients' ability to experience rich and diverse emotional experiences across different disorders. The theoretical and clinical implications of these findings are discussed.

Validity of evoked potential as biomarker for predicting early neural function changes after thoracic spinal decompression surgery in patients with neurological deficits.

OBJECTIVE: To evaluate the validity of intraoperative evoked potential (EP) including motor evoked potential (MEP) and somatosensory evoked potentials (SEP) as a biomarker for predicting neural function changes after thoracic spinal decompression (TSD) surgery. METHOD: A consecutive series of 336 TSD surgeries were reviewed between 2010 and 2021 from four spine center. All patients with TSD were divided into 3 groups according to different intraoperative EP results: group 1, EP alerts; group 2, no obvious EP deterioration; group 3, EP improvement compared with baselines. The lower limb Japanese Orthopedic Association (JOA) scores (as well as early and long-term JOA recovery rate) were utilized to quantitatively assess pre- and postoperative neural function change. RESULTS: Among the 3 subgroups according to the different EP changes, the early JOA recovery rate (RR%) in the EP improvement group was significantly better than the other two groups (51.3 ± 58.6* vs. 27.5 ± 31.2 and 33.3 ± 43.1; p < 0.01) after 3-month follow-up. The mean MEP and SEP amplitude were from 116 ± 57 µV to 347 ± 71 µV (p < 0.01) and from 1.86 ± 0.24 µV to 2.65 ± 0.29 µV (p < 0.01) between spinal cord pre-decompression and post-decompression. Moreover, multivariate logistic regression analysis revealed that risk factors of EP improvement were duration of symptom (p < 0.001, OR 10.9) and Preop. neurologic deficit degree (p = 0.013, OR 7.46). CONCLUSION: The intraoperative EP can predict postoperative neural function changes as a biomarker during TSD. Patient with EP improvement probably has better prognosis for early neural function recovery. The duration of symptom and preoperative neurologic deficit degree may be related to intraoperative EP improvement.

Unleashing diversity through flexibility: The evolutionary journey of sex chromosomes in amphibians and reptiles.

Sex determination systems have greatly diversified between amphibians and reptiles, with such as the different sex chromosome compositions within a single species and transition between temperature-dependent sex determination (TSD) and genetic sex determination (GSD). In most sex chromosome studies on amphibians and reptiles, the whole-genome sequence of Xenopous tropicalis and chicken have been used as references to compare the chromosome homology of sex chromosomes among each of these taxonomic groups, respectively. In the present study, we reviewed existing reports on sex chromosomes, including karyotypes, in amphibians and reptiles. Furthermore, we compared the identified genetic linkages of sex chromosomes in amphibians and reptiles with the chicken genome as a reference, which is believed to resemble the ancestral tetrapod karyotype. Our findings revealed that sex chromosomes in amphibians are derived from genetic linkages homologous to various chicken chromosomes, even among several frogs within single families, such as Ranidae and Pipidae. In contrast, sex chromosomes in reptiles exhibit conserved genetic linkages with chicken chromosomes, not only across most species within a single family, but also within closely related families. The diversity of sex chromosomes in amphibians and reptiles may be attributed to the flexibility of their sex determination systems, including the ease of sex reversal in these animals.

Visible Light and Triselenium Dicyanide (TSD): New Horizons in the Synthesis of Organic Selenocyanates.

Herein, we describe a new method for the synthesis of α-carbonyl selenocyanates by reacting triselenium dicyanide (TSD) and styrenes under blue light irradiation and O2 atmosphere. The reactions are triggered by the formation of Se-centered radical species, followed by the addition/oxidation of the styrene π-bond. α-Carbonyl selenocyanates and α-hydroxy selenocyanates were obtained in moderate to excellent yields from aryl- and alkyl-substituted alkenes, respectively. It was demonstrated that α-carbonyl selenocyanates could be used as a synthetic platform in a multicomponent reaction strategy to prepare 2-phenylimidazo[1,2-a]pyridine derivatives, which were evaluated for their photophysical properties. Overall, this new method provides a useful tool for synthesizing α-carbonyl selenocyanates, and demonstrates their potential for use in the synthesis of other compounds, thus giving new synthetic opportunities to construct organic selenocyanate compounds.

Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.).

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor ß superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

Higher temperatures directly increase germ cell number, promoting feminization of red-eared slider turtles.

In many reptile species, gonadal sex is affected by environmental temperature during a critical period of embryonic development-a process known as temperature-dependent sex determination (TSD).1 The oviparous red-eared slider turtle, Trachemys scripta, has a warm-female/cool-male TSD system and is among the best-studied members of this group.2 When incubated at low temperatures, the somatic cells of the bipotential gonad differentiate into Sertoli cells, the support cells of the testis, whereas at high temperatures, they differentiate into granulosa cells, the support cells of the ovary.3 Here, we report the unexpected finding that temperature independently affects the number of primordial germ cells (GCs) in the embryonic gonad at a time before somatic cell differentiation has initiated. Specifically, embryos incubated at higher, female-inducing temperatures have more GCs than those incubated at the male-inducing temperature. Furthermore, elimination of GCs in embryos incubating at intermediate temperatures results in a strong shift toward male-biased sex ratios. This is the first evidence that temperature affects GC number and the first evidence that GC number influences sex determination in amniotes. This observation has two important implications. First, it supports a new model in which temperature can impact sex determination in incremental ways through multiple cell types. Second, the findings have important implications for a major unresolved question in the fields of ecology and evolutionary biology-the adaptive significance of TSD. We suggest that linking high GC number with female development improves female reproductive potential and provides an adaptive advantage for TSD.

Ribosomal Intergenic Spacers Are Filled with Transposon Remnants.

Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly conserved coding genes separated by rapidly evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one ribosomal RNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently used part of the genome.

Optical profilometry for forensic bloodstain imaging.

Understanding the physical, chemical and biological changes that occur during the drying of a bloodstain is important in many aspects of forensic science including bloodstain pattern analysis and time since deposition estimation. This research assesses the use of optical profilometry to analyze changes in the surface morphology of degrading bloodstains created using three different volumes (4, 11, and 20 µL) up to 4 weeks after deposition. We analyzed six surface characteristics, including surface average roughness, kurtosis, skewness, maximum height, number of cracks and pits, and height distributions from the topographical scans obtained from bloodstains. Full and partial optical profiles were obtained to examine long-term (minimum of 1.5-h intervals) and short-term (5-min intervals) changes. The majority of the changes in surface characteristics occurred within the first 35 min after bloodstain deposition, in agreement with current research in bloodstain drying. Optical profilometry is a nondestructive and efficient method to obtain surface profiles of bloodstains, and can be integrated easily into additional research workflows including but not limited to time since deposition estimation. Optical profilometry is a non-contact tool to scan bloodstains in ambient conditions Drying phases are observable in small drip bloodstains Significant surface morphology changes occur within 35 min after deposition.

Biochemical and mutational analyses of HEXA in a cohort of Egyptian patients with infantile Tay-Sachs disease. Expansion of the mutation spectrum.

BACKGROUND: Tay-Sachs disease (TSD), an autosomal recessively inherited neurodegenerative lysosomal storage disease, reported worldwide with a high incidence among population of Eastern European and Ashkenazi Jewish descent. Mutations in the alpha subunit of HEXA that encodes for the ß-hexosaminidase-A lead to deficient enzyme activity and TSD phenotype. This study is the first to highlight the HEXA sequence variations spectrum in a cohort of Egyptian patients with infantile TSD. RESULTS: This study involved 13 Egyptian infant/children patients presented with the infantile form of TSD, ten of the 13 patients were born to consanguineous marriages. ß-hexosaminidase-A enzyme activity was markedly reduced in the 13 patients with a mean activity of 3 µmol/L/h ± 1.56. Sanger sequencing of the HEXA' coding regions and splicing junctions enabled a detection rate of ~ 62% (8/13) in our patients revealing the molecular defects in eight patients; six homozygous-mutant children (five of them were the product of consanguineous marriages) and two patients showed their mutant alleles in heterozygous genotypes, while no disease-causing mutation was identified in the remaining patients. Regulatory intragenic mutations or del/dup may underlie the molecular defect in those patients showing no relevant pathogenic sequencing variants or in the two patients with a heterozygous genotype of the mutant allele. This research identified three novel, likely pathogenic variants in association with the TSD phenotype; two missense, c.920A > C (E307A) and c.952C > G (H318D) in exon 8, and a single base deletion c.484delG causing a frameshift E162Rfs*37 (p.Glu162ArgfsTer37) in exon 5. Three recurrent disease-causing missense mutations; c.1495C > T (R499C), c.1511G > A(R504H), and c.1510C > T(R504C) in exon 13 were identified in five of the eight patients. None of the variants was detected in 50 healthy Egyptians' DNA. Five variants, likely benign or of uncertain significance, S3T, I436V, E506E, and T2T, in exons 1, 11,13, & 1 were detected in our study. CONCLUSIONS: For the proper diagnostics, genetic counseling, and primary prevention, our study stresses the important role of Next Generation Sequencing approaches in delineating the molecular defect in TSD-candidate patients that showed negative Sanger sequencing or a heterozygous mutant allele in their genetic testing results. Interestingly, the three recurrent TSD associated mutations were clustered on chromosome 13 and accounted for 38% of the HEXA mutations detected in this study. This suggested exon 13 as the first candidate for sequencing screening in Egyptian patients with infantile TSD. Larger studies involving our regional population are recommended, hence unique disease associated pathogenic variations could be identified.

BloodNet: An attention-based deep network for accurate, efficient, and costless bloodstain time since deposition inference.

The time since deposition (TSD) of a bloodstain, i.e., the time of a bloodstain formation is an essential piece of biological evidence in crime scene investigation. The practical usage of some existing microscopic methods (e.g., spectroscopy or RNA analysis technology) is limited, as their performance strongly relies on high-end instrumentation and/or rigorous laboratory conditions. This paper presents a practically applicable deep learning-based method (i.e., BloodNet) for efficient, accurate, and costless TSD inference from a macroscopic view, i.e., by using easily accessible bloodstain photos. To this end, we established a benchmark database containing around 50,000 photos of bloodstains with varying TSDs. Capitalizing on such a large-scale database, BloodNet adopted attention mechanisms to learn from relatively high-resolution input images the localized fine-grained feature representations that were highly discriminative between different TSD periods. Also, the visual analysis of the learned deep networks based on the Smooth Grad-CAM tool demonstrated that our BloodNet can stably capture the unique local patterns of bloodstains with specific TSDs, suggesting the efficacy of the utilized attention mechanism in learning fine-grained representations for TSD inference. As a paired study for BloodNet, we further conducted a microscopic analysis using Raman spectroscopic data and a machine learning method based on Bayesian optimization. Although the experimental results show that such a new microscopic-level approach outperformed the state-of-the-art by a large margin, its inference accuracy is significantly lower than BloodNet, which further justifies the efficacy of deep learning techniques in the challenging task of bloodstain TSD inference. Our code is publically accessible via https://github.com/shenxiaochenn/BloodNet. Our datasets and pre-trained models can be freely accessed via https://figshare.com/articles/dataset/21291825.

Evaluation of the mother's anxiety and child's fear in two different age groups in the child's first and second dental visit.

The anxiety of the mother influences the child's behaviour in a dental setting. Objectives: The study aimed at evaluating the mother's anxiety and a child's fear of first and second dental visits in two different age groups. Study Design: The cross-sectional study design consisted of a total of 100 mother-child pairs attending Pediatric Dental clinics was included in the study. Group I consisted of 50 mother-child pairs of 6-8 years of age. Group II consisted of another 50 mother-child pairs between 12-15 years of age. Short Form of the Dental Subscale of the Children's Fear Survey Schedule (DFSS-SF) was administered to the child. Corah's Dental anxiety scale was administered to the mother. The Tell-Show-Do (TSD) technique was used in all children before the treatment. Statistical Analysis Used: SPSS software 21 was used for descriptive and inferential statistics. Pearson's correlation coefficient was used for bivariate correlation between variables in the study. Results: The anxiety level of mothers on both appointment days in both age groups was found to be highly significant. The correlation of maternal anxiety to the gender of the child in both groups was found to be highly significant. Conclusion: The fear of dental treatment was commonly found in children irrespective of gender in both age groups. The TSD technique was found to reduce fear in the subsequent appointment.

Long-Term Resilience of Primary Sex Ratios in a Species with Temperature-Dependent Sex Determination after Decades of Climate Warming.

AbstractSpecies with environmental sex determination (ESD) have persisted through deep time, despite massive environmental perturbation in the geological record. Understanding how species with temperature-dependent sex determination (TSD), a type of ESD, persist through climate change is particularly timely given the current climate crisis, as highly biased sex ratios and extinction are predicted. Since 1982, we have studied primary sex ratios of a reptile with TSD (Chelydra serpentina). Primary sex ratios remained unchanged over time, despite warming in the environment. Resilience of the primary sex ratio occurred via a portfolio effect, realized through remarkable intra-annual variation in nest-level sex ratios, leading to a relatively consistent mean annual sex ratio. Intra-annual variation in nest-level sex ratios was related to variation in egg burial depth coupled with large clutch sizes, creating thermal gradients in the nest and promoting mixed-sex clutches. Furthermore, both locally and globally, sustained increases in nighttime air temperature contribute more to warming than increases in daily maximum temperature, but development rate was affected more strongly by maximum daily air temperature, conferring additional resilience to overall warming. Our study suggests that some TSD species may be resilient to warming and provides an example of how ESD may persist under environmental change.

Evolution and dosage compensation of nucleolar organizing regions (NORs) mediated by mobile elements in turtles with female (ZZ/ZW) but not with male (XX/XY) heterogamety.

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.

Therapeutic Strategies For Tay-Sachs Disease.

Tay-Sachs disease (TSD) is an autosomal recessive disease that features progressive neurodegenerative presentations. It affects one in 100,000 live births. Currently, there is no approved therapy or cure. This review summarizes multiple drug development strategies for TSD, including enzyme replacement therapy, pharmaceutical chaperone therapy, substrate reduction therapy, gene therapy, and hematopoietic stem cell replacement therapy. In vitro and in vivo systems are described to assess the efficacy of the aforementioned therapeutic strategies. Furthermore, we discuss using MALDI mass spectrometry to perform a high throughput screen of compound libraries. This enables discovery of compounds that reduce GM2 and can lead to further development of a TSD therapy.

Estimating the time since deposition (TsD) in saliva stains using temporal changes in microbial markers.

Determining the time since deposition (TsD) of traces could be helpful in the investigation of criminal offenses. However, there are no reliable markers and models available for the inference of short-term TsD. The goal of this study was to investigate the potential of the succession pattern of human salivary microbial communities to serve as an efficiency TsD prediction tool in the resolution of the forensic cases. Saliva stains exposed to indoor conditions up to 20 days were collected and analyzed by 16S rRNA profiling using high-throughput sequencing technique. Noticeable differences in microbial composition were observed between different time points, and the indoor exposure time of saliva stains were inversely correlated with alpha diversity estimates across the measured time period. The sequencing results were used to identify TsD-dependent bacterial indicators to regress a generalized random forest model, resulting in a mean absolute deviation (MAD) of 1.41 days. Furthermore, a simplified TsD predictive model was also developed utilizing Enhydrobacter, Paenisporosarcina, and Janthinobacterium by quantitative PCR (qPCR) with a MAD of 1.32 days, and then forensic practice assessment were also performed by using mock samples with a MAD of 3.53 days. In conclusion, this study revealed significant changes in salivary microbial abundance as the prolongation of TsD. It demonstrated that the microbial biomarkers could be invoked as a "clock" for TsD estimation in human dried saliva stains.

Quantitative Determination of Four Potential Genotoxic Impurities in the Active Pharmaceutical Ingredients in TSD-1 Using UPLC-MS/MS.

A novel method of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of four potential genotoxic impurities (PGIs) in the active pharmaceutical ingredients of TSD-1, a novel P2Y12 receptor antagonist. Four PGIs were named, 4-nitrobenzenesulfonic acid, methyl 4-nitrobenzenesulfonate, ethyl 4-nitrobenzenesulfonate, and isopropyl 4-nitrobenzenesulfonate. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying four PGIs at 15.0 ppm in samples of 0.5 mg/mL concentration. This validated approach presented very low limits (0.1512−0.3897 ng/mL), excellent linearity (coefficients > 0.9900), and a satisfactory recovery range (94.9−115.5%). The method was sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity, and robustness and, thus, has high practicality in the pharmaceutical quality control of TSD-1.