Background/Objectives: The goal of this study was to characterize changes in autofluorescence of epithelial cells obtained from saliva stains that occur with time and investigate the potential for these changes to serve as time-since-deposition (TSD) signatures for this sample type. Methods: Saliva from 50 individuals was used to create 208 deposits that were aged between one day and nine months. Autofluorescence profiles of individual cells were obtained from each sample using imaging flow cytometry (IFC) and analyzed across nine different emission channels ranging between 435 nm and 800 nm. Results: Results showed strong evidence for linear increases in autofluorescence intensity when epithelial cells from a single donor deposit were measured over time (12 of 14 donors r >= 0.9). When autofluorescence profiles from all 50 donors were combined into a single time series, variation in autofluorescence intensity was observed between individual deposits with the same TSD. This inter-contributor variation decreased the overall strength of the linear relationship (r = 0.83) and yielded residual errors of similar to 8 days for samples that were actually 1 day old and similar to 82 days for samples that were over 180 days old using a linear regression model. Although this approach may not currently be amenable to estimating TSD to the day with high accuracy, clear, non-overlapping differences in autofluorescence intensity were still observed between certain time intervals, e.g., saliva deposits that were aged for 1 day compared to saliva deposits that were aged for more than 120 days. Conclusions: This suggests that cellular autofluorescence signatures have the potential to be probative when hypotheses for sample deposition involve disparate time intervals or as a screening tool for identifying which samples are most likely relevant to the crime in question based on their deposition time.

Animal cruelty investigations can be hampered by not knowing when a biological sample was deposited at a crime scene, i.e., the sample’s time-since-deposition (TSD). The goal of this study was to characterize a new signature system for estimating the TSD of canine blood samples based on changes in the autofluorescence spectra of cell populations that occur over time. The results showed that the intensity of cellular autofluorescence measured in two detector channels (523/30 nm and 695/50 nm; 488 nm excitation) could clearly differentiate older blood deposits (i.e., samples with a TSD of either 6 months or 1 year) from younger samples that had a TSD of less than three months. Further, cellular autofluorescence was strongly correlated with time when the sample TSD was between one day and three months, with correlation coefficients ranging between 0.83 and 0.91. A multiple linear regression model based on autofluorescence variation was tested on mock casework samples and yielded residual errors between 2 days and 35 days, with the lowest residuals observed in samples that have a TSD less than 6 days. This information can provide probative leads during an investigation of an illicit canine fighting event or an individual injury and also allow animal cruelty agencies to triage blood samples based on their TSD for downstream analyses.

Background New methods for processing ‘touch’ or trace biological samples is an ongoing priority for forensic caseworking laboratories. These samples often contain materials from multiple individuals in varying quantities and/or degrees of degradation. Rapid characterization of cellular material before DNA profiling can allow laboratories to screen samples for the presence of multiple contributors or the amount of biological material present. Methods This dataset contains autofluorescence and morphological profiles of epidermal cell populations analyzed using Imaging Flow Cytometry. The epidermal samples were aged for varying amounts of time prior to analysis. Multiple samples from the same individual were also collected to assess profile variations within and across the contributors. Conclusions This data set may be used to investigate variability in epidermal cell populations from different individuals and potential forensic signatures contained within the non-genetic components that comprise touch biological evidence.

Determining when DNA recovered from a crime scene transferred from its biological source, i.e., a sample's 'time-since-deposition' (TSD), can provide critical context for biological evidence. Yet, there remains no analytical techniques for TSD that are validated for forensic casework. In this study, we investigate whether morphological and autofluorescence measurements of forensically-relevant cell populations generated with Imaging Flow Cytometry (IFC) can be used to predict the TSD of 'touch' or trace biological samples. To this end, three different prediction frameworks for estimating the number of day(s) for TSD were evaluated: the elastic net, gradient boosting machines (GBM), and generalized linear mixed model (GLMM) LASSO. Additionally, we transformed these continuous predictions into a series of binary classifiers to evaluate the potential utility for forensic casework. Results showed that GBM and GLMM-LASSO showed the highest accuracy, with mean absolute error estimates in a hold-out test set of 29 and 21 days, respectively. Binary classifiers for these models correctly binned 94-96% and 98-99% of the age estimates as over/under 7 or 180 days, respectively. This suggests that predicted TSD using IFC measurements coupled to one or, possibly, a combination binary classification decision rules, may provide probative information for trace biological samples encountered during forensic casework.

This work explores morphological and autofluorescence differences between vaginal and epidermal cells detectable through Imaging Flow Cytometry (IFC), a non-destructive, high-throughput technique. These differences were used to build a predictive framework for classifying unknown cells as originating from vaginal or epidermal tissue, which was tested on hand swabbings with and without digital penetration. Many more cells possessing a vaginal signature (median posterior probability ≥0.90) were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize/eliminate false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework.

Although there are many techniques to detect pathogenic bacteria, most of them are only suited for in vitro diagnostics. We report a urinary catheter-based colorimetric sensor for potential on-body detection of E. coli, the most prevalent bacterial species in urinary tract infections associated with the use of urinary catheters. In urine, indole is secreted by E. coli and reacts with a nitrosating agent incorporated in a silicone catheter. A red dimeric product, indoxyl red, is generated within silicone rubber to allow for color-based indole sensing with high sensitivity, linearity, and specificity. This reaction is initiated by the nitrosation reaction of indole at its C-3 position via reagents like sodium nitrite or S-nitroso-N-acetyl-penicillamine under aerobic conditions. The generated 3-nitrosoindole undergoes tautomerization, dimerization, and deoximation to form indoxyl red with high absorbance at 537 nm. In contrast to other indole sensors, the presented method can be applied in real catheters to detect indole and E. coli in biofluids such as urine. The is because (1) S-nitroso-N-acetyl-penicillamine, the nitrosating agent, can be impregnated into silicone elastomers, (2) indole from urine is extracted into silicone due to its hydrophobicity, and (3) the high acidity and oxygen solubility of silicone facilitates the sensing reaction within the silicone matrix. This silicone-based colorimetric sensor clearly differentiates E. coli below and above 105 CFU/mL, which is the threshold concentration of bacteriuria. We expect that early diagnosis of urinary tract infections using the naked eye is possible by functionalizing an exposed section of urinary catheters with the proposed molecular probe.

A significant proportion of casework analyzed by forensic science laboratories is often "touch" or trace forensic DNA evidence, which is deposited through physical contact and is comprised of sloughed epidermal cells. These samples can be challenging to analyze due to low DNA concentrations, frequent degradation, and the presence of cells from multiple individuals in the same sample. To address these challenges, we investigated a new approach for characterizing trace evidence prior to DNA profiling that labels epidermal cells with antibody probes targeting hormone molecules testosterone and dihydrotestosterone (DHT). The goal was to test whether cell populations derived from separate individuals showed different binding efficiencies to hormone probes and, thus, could be used to detect the presence of multiple cell populations. Additionally, we investigated whether antibody probes could be used to isolate contributor cell populations from an epidermal cell mixture and facilitate deconvolution of mixed DNA profiles recovered from touch/trace evidence. Results showed that cell populations from some individuals could differentiated in trace samples based on fluorescence histograms following probe labeling. However, certain pairs of contributors showed largely or completely overlapping histogram profiles and could not be resolved. Preliminary efforts to separate cell populations that could be differentiated with hormone probes with fluorescence-activated cell sorting (FACS) coupled to DNA profiling and probabilistic modeling indicated that it is possible to enrich contributor cell populations from touch/trace samples and produce more probative DNA profiles compared to the original mixture sample. The variability in labeling, differentiation, and physical separation of cell populations may be impacted by similarities in biochemical profiles across some contributors as well as imbalance of contributor DNA quantities in certain mixtures as is typical in casework involving touch/trace evidence. Ultimately, screening and separation of trace DNA samples with this approach may be presumptive and constrained by sample-specific parameters of the original mixture.

With the increase in sensitivity of DNA profiling, questions about how and when the DNA was deposited have become a driving issue in forensic cases. To address this, we propose a novel method to determine time since deposition of trace DNA samples based on morphological and autofluorescence properties of individual epithelial cells which can change as the sample ages. To develop this signature, a series of trace DNA samples were generated by contact/handling a substrate and then allowed to age anywhere between one day and more than one year prior to collection. Imaging flow cytometry (IFC) was then used to characterize the morphology and autofluorescence profiles of individual cells within each sample followed by multivariate modelling and predictive classification. Results: showed that epithelial cell populations could be classified with high accuracy (-90%) into one of three time-since-deposition groups: < 1 week, between 1 week and 2 months, and > 2months. Differences across age groups were largely driven by decreases in brightfield contrast and increases in the intensity of autofluorescence. To further test this approach for forensic casework, 47 individual donor cell populations spanning each time deposition group were classified blindly against the remaining data set. Samples containing at least 75 cells and a posterior probability greater than 0.90 showed classification accuracies -95%. Accuracies for individual time groups were 97% (<1 week), 92% (1week-2months), 98% (>2 months) with an average posterior probability for all time groups -0.96. This indicates that autofluorescence and morphological analyses may provide probative information regarding time since deposition for many types of trace DNA samples in forensic casework.

The atomic force microscope (AFM) has found its way to the arsenal of tools available to the forensic practitioner for the analysis of samples at the nano and microscales. As a non-destructive probing tool that requires minimal sample preparation, the AFM is very attractive, particularly in the case of minimal or precious sample. To date, the use of the AFM has primarily been in the arena of imaging where it has been complementary to other microscopic examination tools. Forensic applications in the visual examination of evidence such as blood stains, questioned documents, and hair samples have been reported. While a number of reviews have focused on the use of AFM as an imaging tool for forensic analyses, here we not only discuss these works, but also point to a versatile enhancement in the capabilities of this nanoscale tool - namely its use for force spectroscopy. In this mode, the AFM can determine elastic moduli, adhesion forces, energy dissipation, and the interaction forces between cognate ligands, that can be spatially mapped to provide a unique spatial visualization of properties. Our goals in this review are to provide a context for this capability of the AFM, explain its workings, cover some exemplary works pertaining to forensic sciences, and present a critical analysis on the advantages and disadvantages of this modality. Equipped with this high-resolution tool, imaging and biophysical analysis by the AFM can provide a unique complement to other tools available to the researcher for the analysis and characterization of forensic evidence.

Controlled release of drugs from medical implants is an effective approach to reducing foreign body reactions and infections. We report here on a one-step 3D printing strategy to create drug-eluting polymer devices with a drug-loaded bulk and a drug-free coating. The spontaneously formed drug-free coating dramatically reduces the surface roughness of the implantable devices and serves as a protective layer to suppress the burst release of drugs. A high viscosity liquid silicone that can be extruded based on its shear-thinning property and quickly vulcanize upon exposure to ambient moisture is used as the ink for 3D printing. S-Nitrosothiol type nitric oxide (NO) donors in their crystalline forms are selected as model drugs because of the potent antimicrobial, antithrombotic, and anti-inflammatory properties of NO. Direct ink writing of the homogenized polymer-drug mixtures generates rough and ill-defined device surfaces because of the exposed S-nitrosothiol microparticles. When a low-viscosity silicone (polydimethylsiloxane) is added into the ink, this silicone diffuses outward upon deposition to form a drug-free outermost layer without compromising the integrity of the printed structures. S-Nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP) embedded in the printed silicone matrix releases NO under physiological conditions from days to about one month. The microsized drug crystals are well-preserved in the ink preparation and printing processes, which is one reason for the sustained NO release. Biofilm and cytotoxicity experiments confirmed the antibacterial property and safety of the printed NO-releasing devices. This additive manufacturing platform does not require dissolution of drugs and involves no thermal or UV processes and, therefore, offers unique opportunities to produce drug-eluting silicone devices in a customized manner.

The goal of this study was to characterize the reproducibility of extracellular and cell pellet associated DNA yields recovered from handled substrates. Results showed that extracellular DNA yields were extremely variable between contributors—ranging between 0 and >10ng—and tended to dwarf cell pellet yields, which varied between 0 and ∼230 pg. DNA yields across multiple samples from the same contributor on different days showed similar levels of variability in both DNA fractions, indicating that extracellular DNA yield is largely influenced by extrinsic and/or environmental factors and is not a contributor-specific attribute. Microscopic surveys of cells from the pellet fraction as well as fingerprints from the same contributor samples were conducted following treatment with fluorescent DNA stain. Nearly all imaged cells exhibited diffuse fluorescence across the cell without discernable evidence of nuclei. This is consistent with the limited nature of DNA recovery from the pellet fraction and the prevalence of extracellular DNA in these samples.

This paper presents a strategy for an unsupervised workflow for identifying epithelial cells in microscopic images and characterizing their morphological and/or optical properties. The proposed method can be used on cells that have been stained with fluorescent dyes and imaged using conventional optical microscopes. The workflow was tested on cell populations that were imaged directly on touch/contact surfaces and stained with nucleic acid dyes to visualize genetic content. Our results show that this approach could be a useful strategy for characterizing differences in staining efficiency and/or morphological properties of individual cells or aggregate populations within a biological sample. Further, they can potentially reduce the laborious nature of microscopic analysis and increase throughput and reproducibility of similar studies.

Journal of Forensic SciencesVolume 66, Issue 1 p. 424-424 Book Review Review of: Microbial forensics, 3rd ed. Christopher J. Ehrhardt PhD, Corresponding Author Christopher J. Ehrhardt PhD cehrhardt@vcu.edu Department of Forensic Science, Virginia Commonwealth University, Richmond, Virginia, USASearch for more papers by this author Christopher J. Ehrhardt PhD, Corresponding Author Christopher J. Ehrhardt PhD cehrhardt@vcu.edu Department of Forensic Science, Virginia Commonwealth University, Richmond, Virginia, USASearch for more papers by this author First published: 21 October 2020 https://doi.org/10.1111/1556-4029.14598Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume66, Issue1January 2021Pages 424-424 RelatedInformation

The persistence of bacterial pathogens within environmental matrices plays an important role in the epidemiology of diseases, as well as impacts biosurveillance strategies. However, the adaptation potentials, mechanisms for survival, and ecological interactions of pathogenic bacteria such as Yersinia pestis are largely uncharacterized owing to the difficulty of profiling their phenotypic signatures. In this report, we describe studies on Y. pestis organisms cultured within soil matrices, which are among the most important reservoirs for their propagation. Morphological (nanoscale) and phenotypic analysis are presented at the single cell level conducted using Atomic Force Microscopy (AFM), coupled with biochemical profiles of bulk populations using Fatty Acid Methyl Ester Profiling (FAME). These studies are facilitated by a novel, customizable, 3D printed diffusion chamber that allows for control of the external environment and easy harvesting of cells. The results show that incubation within soil matrices lead to reduction of cell size and an increase in surface hydrophobicity. FAME profiles indicate shifts in unsaturated fatty acid compositions, while other fatty acid components of the phospholipid membrane or surface lipids remained consistent across culturing conditions, suggesting that phenotypic shifts may be driven by non-lipid components of Y. pestis.

Negatively charged lipopolysaccharide (LPS), a major endotoxin and component of the outer membrane of several Gram-negative bacteria, provides a useful biomarker for the indirect detection of these pathogens. For instance, Escherichia coli (E. coli) is a pathogenic bacterium that causes infections in almost all age groups, and has been implicated in food and water contamination. Current diagnostic and detection methods tend to be labor-intensive or expensive, necessitating the need for an easy, sensitive, rapid, and low-cost method. We report on the synthesis and use of positively charged chitosan stabilized silver nanoparticles (Chi-AgNPs) as a sensitive electrochemical nanobiosensor for the detection of LPS. Chi-AgNPs were synthesized through a facile, single step protocol, and characterized for size, charge, and morphology. Glassy carbon electrodes modified with Chi-AgNPs resulted in an enhancement of signal in the presence of both LPS and E. coli. Detection was accomplished over a large concentration range (several orders of magnitude) of 0.001–100 ng/mL and 10–107 CFU/mL. The biosensors can reliably detect LPS and E. coli at very low concentrations. Chi-AgNPs have potential as low cost, sensitive nanobiosensors for Gram-negative bacteria due to strong electrostatic interaction with LPS present in their outer membranes.

Nanoscale analysis of extracellular DNA (eDNA) that is present on the surface of cells in trace biological samples can provide insight into the understanding of DNA transfer through touch, and thereby, the role of eDNA is a biologically and forensically relevant phenomenon. While various bulk scale tools and DNA analysis can be used to quantitatively obtain this information, obtaining a three dimensional (3D) visualization of the eDNA can provide a unique look into the spatial and temporal dynamics at the cellular level. In this study, we show how atomic force microscopy (AFM) can be integrated with optical microscopy to visualize the distribution of surface associate eDNA at a single cell level. Using a nucleic acid fluorophore such as Diamond™ Dye, the surface eDNA can be observed and quantified using fluorescence microscopy. This informational channel can then be overlaid with surface topography and cellular elasticity to provide structural visualization. Finally, chemical force spectroscopy can be used to obtain the distribution of surface-associated eDNA on the cell surface at the molecular level. Such integrated techniques can enhance understanding of the biological role of eDNA, and can also be potentially valuable for investigating challenging trace samples, containing very few cells for various analyses.

Multiple drug resistant (MDR) has become a major issue in developing countries. MDR bacterial infections lead to significant increase in morbidity, mortality and cost of prolonged treatments. Therefore, designing of strategies for improving the antimicrobial potential of the therapeutic agents are highly required. Metal organic frameworks (MOFs) are highly tunable hybrid material, consist of metal ions linked together by organic bridging ligands have been used as an efficient drug delivery carrier because of their biodegradability, low toxicity and structure integrity upon loading and functionalizing process. Current study was based on the synthesis of chitosan coated MOFs with enhanced contact with S. aureus cell surface. Chitosan is deacetylated derivative of chitin and capable for non-bonding interaction with negatively charged bacterial cell leading to enhanced contact of MOFs with S. aureus. Chitosan coated MOFs were characterized with various techniques such as atomic force microscopy, scanning electron microscopy, DLS, FT-IR, TGA, DSC and Powder X-ray diffraction. They were also studied for their efficacy on resistant S. aureus, results revealed that Vancomycin bactericidal activity significantly increased upon loading in chitosan coated MOFs and caused increased inhibition of resistant S. aureus. AFM analysis of S. aureus strains clearly revealed complete distortion of morphology by treating with chitosan modified drug loaded MOFs. Findings of the current study suggest the potential of chitosan coated MOFs for reversing bacterial resistance against Vancomycin and provide new perspectives for improved antibiotic therapy of infections associated with MDR.