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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.

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.

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&ndash, 100 ng/mL and 10&ndash, 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.

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.

Accurate blood detection is a primary concern for forensic scientists, especially in highly compromised situations. In this study, blood was added to wood blocks and subjected to a variety of fire treatments: the absence or presence of accelerant, burn time (1, 3, or 5 min), and extinguishment method (smothering or dousing with water). Burned blocks were given a qualitative burn score, followed by removal of half of the char from each block and subsequent testing of each half for blood using luminol (13% positive; n = 96), Bluestar® Forensic Magnum (5.2% positive; n = 96), and combined phenolphthalein tetramethylbenzidine test (0% positive; n = 192). Luminol and Bluestar® Forensic Magnum performed similarly, both outperforming PTMB. Additionally, positive results were more likely from samples that were smothered, had a low burn score, and had more concentrated blood solutions (neat or 1:2). Overall, it is extremely unlikely that blood would be detected on combustible substrates exposed to direct fire.

Forensic samples comprised of cell populations from multiple contributors often yield DNA profiles that can be extremely challenging to interpret. This frequently results in decreased statistical strength of an individual’s association to the mixture and the loss of probative data. The purpose of this study was to test a front-end cell separation workflow on complex mixtures containing as many as five contributors. Our approach involved selectively labelling certain cell populations in dried whole blood mixture samples with fluorescently labeled antibody probe targeting the HLA-A*02 allele, separating the mixture using Fluorescence Activated Cell Sorting (FACS) into two fractions that are enriched in A*02 positive and A*02 negative cells, and then generating DNA profiles for each fraction. We then tested whether antibody labelling and cell sorting effectively reduced the complexity of the original cell mixture by analyzing STR profiles quantitatively using the probabilistic modeling software, TrueAllele® Casework. Results showed that antibody labelling and FACS separation of target populations yielded simplified STR profiles that could be more easily interpreted using conventional procedures. Additionally, TrueAllele® analysis of STR profiles from sorted cell fractions increased statistical strength for the association of most of the original contributors interpreted from the original mixtures.

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650–670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor’s contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650–670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical AbstractConceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0364-0) contains supplementary material, which is available to authorized users.

“Touch” or trace DNA evidence represent a significant proportion of samples analyzed by forensic science laboratories. Because these samples frequently contain multiple contributors and are often challenging to analyze due to low DNA concentrations and frequent degradation, front end techniques to simplify the mixture prior to DNA profiling could significantly impact case processing and enhance success rates. The goal of this study was to investigate whether targeting hormone molecules within the cell with antibody probes could be used to selectively label and then physically isolate contributor cell populations in trace biological samples. The separation of male and female cells into distinct fractions could reduce the complexity of the mixture prior to DNA profiling. To accomplish this, we first tested the specificity of fluorescently labelled anti-testosterone and anti-dihydrotestosterone antibody probes to epidermal cells from both male and female individuals. Results show that male and female cell populations can be effectively labeled using anti-testosterone and anti-dihydrotestosterone antibody probes and that distinct differences in binding efficiency and resulting median fluorescence of cell populations were observed between several individuals. These differences were then used to design sorting criteria for physically isolating each cell population in two-person epidermal cell mixtures using fluorescence activated cell sorting (FACS). DNA profiling of separated fractions in combination with probabilistic modeling demonstrated that some cell mixtures could be enriched for one contributor in separated cell fractions and yielded statistically more discriminating profiles compared to those generated from the original mixtures. Other mixtures tested showed less evidence of effective cell separation possibly due to a number of factors including imbalance of contributor DNA ratio, intra-sex variation of antibody binding efficiency, and contributions of extracellular or cell-free DNA in the mixture sample. Screening and separation of trace DNA samples with this approach may be presumptive and ultimately constrained by specific parameters of the original mixture, however, antibody binding optimization may mitigate some of these influences.

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.

Gaps in our understanding of the natural ecology and survival mechanisms of pathogenic bacteria in complex microenvironments such as soil typically occur due to the difficulty in characterizing biochemical profiles and morphological characteristics as they exist in environmental samples. Conversely, accurate simulation of the abiotic and biotic chemistries of soil habitats within the laboratory is often a significant challenge. Herein, we present the fabrication of customizable and precisely engineered 3D printed diffusion chambers that can be used to incubate bacterial cultures directly in soil matrices within a controlled laboratory experiment, and study the dynamics between bacterial cells and soil components. As part of the design process, different types of 3D printing materials were evaluated for ease of sterilization, structural integrity throughout the experiment, as well as cost/ease of production. To demonstrate potential applications for environmental studies, the diffusion chamber was used to incubate cultures of Bacillus cereus T-strain and Escherichia coli strain O157 directly in soil matrices. We show that the chamber facilitates diffusion of abiotic/biotic components of the soil with target cells without contamination from in situ microbial communities, while allowing for single cell and ensemble level phenotypic analyses of bacteria cultured with and without soil matrices.

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.

Yersinia pestis, the causative agent of plague, has been responsible for several recurrent, lethal pandemics in history. Currently, it is an important pathogen to study owing to its virulence, adaptation to different environments during transmission, and potential use in bioterrorism. Here, we report on the changes to Y. pestis surfaces in different external microenvironments, specifically culture temperatures (6, 25, and 37 °C). Using nanoscale imaging coupled with functional mapping, we illustrate that changes in the surfaces of the bacterium from a morphological and biochemical standpoint can be analyzed simultaneously using atomic force microscopy. The results from functional mapping, obtained at a single cell level, show that the density of lipopolysaccharide (measured via terminal N-acetylglucosamine) on Y. pestis grown at 37 °C is only slightly higher than cells grown at 25 °C, but nearly three times higher than cells maintained at 6 °C for an extended period of time, thereby demonstrating that adaptations to different environments can be effectively captured using this technique. This nanoscale evaluation provides a new microscopic approach to study nanoscale properties of bacterial pathogens and investigate adaptations to different external environments.

Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 μg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 μg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 μg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

Summary Manipulation of viable Yersinia pestis (etiologic agent of plague) in the laboratory usually necessitates elevated biosafety and biocontainment procedures, even with avirulent or vaccine strains. To facilitate downstream biochemical or physical analyses in a Biosafety Level 1 laboratory environment, effective inactivation without affecting its intrinsic properties is critical. Here, we report on the morphological and biochemical changes to Y. pestis surfaces following four different fixation methods that render the cells nonviable. The results, obtained at the single cell level, demonstrate that methanol inactivation is best able to preserve bacterial morphology and bioactivity, enabling subsequent analysis. This nanoscale evaluation of the effects of inactivation on cell morphology and surface bioactivity may provide a crucial preparatory approach to study virulent pathogens in the lab setting using high-resolution microscopic techniques such as atomic force microscopy.

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.

Computed tomography (CT) scans provide anthropologists with a resource to generate three-dimensional (3D) digital skeletal material to expand quantification methods and build more standardized reference collections. The ability to visualize and manipulate the bone and skin of the face simultaneously in a 3D digital environment introduces a new way for forensic facial approximation practitioners to access and study the face. Craniofacial relationships can be quantified with landmarks or with surface-processing software that can quantify the geometric properties of the entire 3D facial surface. This article describes tools for the generation of dense facial tissue depth maps (FTDMs) using deidentified head CT scans of modern Americans from the Cancer Imaging Archive public repository and the open-source program Meshlab. CT scans of 43 females and 63 males from the archive were segmented and converted to 3D skull and face models using Mimics and exported as stereolithography files. All subsequent processing steps were performed in Meshlab. Heads were transformed to a common orientation and coordinate system using the coordinates of nasion, left orbitale, and left and right porion. Dense FTDMs were generated on hollowed, cropped face shells using the Hausdorff sampling filter. Two new point clouds consisting of the 3D coordinates for both skull and face were colorized on an RGB (red-green-blue) scale from 0.0 (red) to 40.0-mm (blue) depth values and exported as polygon (PLY) file format models with tissue depth values saved in the "vertex quality" field. FTDMs were also split into 1.0-mm increments to facilitate viewing of common depths across all faces. In total, 112 FTDMs were generated for 106 individuals. Minimum depth values ranged from 1.2 mm to 3.4 mm, indicating a common range of starting depths for most faces regardless of weight, as well as common locations for these values over the nasal bones, lateral orbital margins, and forehead superior to the supraorbital border. Maximum depths were found in the buccal region and neck, excluding the nose. Individuals with multiple scans at visibly different weights presented the greatest differences within larger depth areas such as the cheeks and neck, with little to no difference in the thinnest areas. A few individuals with minimum tissue depths at the lateral orbital margins and thicker tissues over the nasal bones (3.0 mm) suggested the potential influence of nasal bone morphology on tissue depths. This study produced visual quantitative representations of the face and skull for forensic facial approximation research and practice that can be further analyzed or interacted with using free software. The presented tools can be applied to preexisting CT scans, traditional or cone beam, adult or subadult individuals, with or without landmarks, and regardless of head orientation, for forensic applications as well as for studies of facial variation and facial growth. In contrast with other facial mapping studies, this method produced both skull and face points based on replicable geometric relationships, producing multiple data outputs that are easily readable with software that is openly accessible.

Accurate blood detection is a primary concern for forensic scientists, especially in highly compromised situations. In this study, blood was added to wood blocks and subjected to a variety of fire treatments: the absence or presence of accelerant, burn time (1, 3, or 5 min), and extinguishment method (smothering or dousing with water). Burned blocks were given a qualitative burn score, followed by removal of half of the char from each block and subsequent testing of each half for blood using luminol (13% positive; n = 96), Bluestar

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, epidermal, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that epidermal cells could be distinguished from vaginal and buccal cells using a discriminant function analysis of IFC measurements with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques.

Establishing the tissue source of epithelial cells within a biological sample is an important capability for forensic laboratories. In this study we used Imaging Flow Cytometry (IFC) to analyze individual cells recovered from buccal, contact epithelial, and vaginal samples that had been dried between 24 hours and more than eight weeks. Measurements capturing the size, shape, and fluorescent properties of cells were collected in an automated manner and then used to build a multivariate statistical framework for differentiating cells based on tissue type. Results showed that cell populations from the three tissue types could be differentiated using a Discriminant Function plot of IFC measurements. Epidermal cells were distinguished from vaginal and buccal cells with an average classification accuracy of ~94%. Ultimately, cellular measurements such as these, which can be obtained non-destructively, may provide probative information for many types of biological samples and complement results from standard genetic profiling techniques.

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.

Some microorganisms have been shown to retain a chemical signature indicative of the medium used for culturing. However, the repeatability of medium-specific chemical signatures has not been demonstrated from samples of microorganisms produced in the same batch or in different batches by the same sporulation protocol. Here, the variation in Raman spectra of bacterial endospores repeatedly prepared by the same procedure is compared to the variation between Raman spectra of spores prepared using different media. Bacillus cereus T strain (BcT) samples were correctly classified according to the medium used to induce sporulation for 100 % of spores grown in a controlled manner by the same scientist using Raman spectroscopy and multivariate data analysis. The proof-of-concept results from BcT spores produced in 12 different sporulation media showed correct classification by medium for 98 % of samples (with 100 % classification accuracy for all but one sporulation medium in this data set). Spectral differences were discerned between spores that had been freshly prepared or freeze-dried and spores that had been frozen; however, the differences did not impact the classification of the sporulation medium. Latent variables reduced the classification accuracy of BcT sporulated in G medium by different scientists using different media lots and stored for different periods of time and requires further study.

Summary Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food-related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterizetheirsurfacemechanically.Theprogressioninelasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.

Craniometric data from computed tomography (CT) head scans of 287 living Americans of three descent groups (African, Asian, European) and both sexes were analyzed for measurement precision. Classification accuracy was estimated by a leave-one-out cross validation, and group means were compared to the Forensic Data Bank (FDB). Landmarks were placed on 3D surface models of the skulls to approximate traditional cranial measurements utilized in sex and ancestry estimations. From repeat measurements by one observer on a subset of skulls (n = 14) reflecting 14 different CT protocols, the least precise landmark was euryon (SD ≤ 4.09 mm) and the least precise distances according to the coefficient of reliability (

We analyzed 21 neat acetone samples from 15 different suppliers to demonstrate the utility of a coupled stable isotope and trace contaminant strategy for distinguishing forensically-relevant samples. By combining these two pieces of orthogonal data we could discriminate all of the acetones that were produced by the 15 different suppliers. Using stable isotope ratios alone, we were able to distinguish 8 acetone samples, while the remaining 13 fell into four clusters with highly similar signatures. Adding trace chemical contaminant information enhanced discrimination to 13 individual acetones with three residual clusters. The acetones within each cluster shared a common manufacturer and might, therefore, not be expected to be resolved. The data presented here demonstrates the power of combining orthogonal data sets to enhance sample fingerprinting and highlights the role disparate data could play in future forensic investigations.

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to differentially label and then sort cell populations from separate contributors in a trace biological mixture. Cell characterizations initially focused on two different protein systems, Human Leukocyte Antigen (HLA) complex and cytokeratin (CK) filaments. Hybridization experiments using pan and allele-specific HLA antibody probes showed that surface antigens on cells transferred from the palmar surface of volunteers are largely unreactive, suggesting that they cannot be used to differentiate cell populations in a touch mixture. Touch samples were also hybridized with the pan-CK probe AE1, which targets CK proteins 10, 14, 15, 16 and 19. Fluorescence levels of AE1 hybridized cells were observed to vary across donors, although these differences were not consistent across all sampling days. We then investigated variations in red autofluorescence profiles (650-670nm) as a potential signature for distinguishing contributor cell populations. Although distinct differences in red autofluorescence profiles were observed ‐‐ with one donor consistently exhibiting higher levels of fluorescence than others ‐‐ some variation was also observed in touch samples collected from the same individual on different days. While this suggests that contributor touch samples cannot be defined by a discrete level of autofluorescence, this attribute may still be a useful means of isolating contributors to some touch mixtures. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two person touch mixture was separated into two fractions via Fluorescence Activated Cell Sorting (FACS) using gating criteria based on intensity of 650-670nm emissions, and then subjected to DNA analysis. STR typing of the sorted fractions provided partial profiles that were consistent with separation of individual contributors from the mixture.

Copper has a long historical role in the arena of materials with antimicrobial properties. Various forms of copper ranging from surfaces to impregnation in textiles and particles, have attracted considerable interest owing to their versatility, potency, chemical stability, and low cost. However, the effects and mechanisms of their antimicrobial action is still unclear. In this study, the effect of copper particles on Escherichia coli was studied at the nanoscale using atomic force microscopy (AFM). Time-lapse AFM images at the single cell level show the morphological changes on live E. coli during antimicrobial treatment, in which for the first time, this process was followed in situ on the same cell over time. AFM-based hydrophobicity mapping further showed that incubating cells with Cu decreased the surface hydrophobicity with an increase of incubation time. Specifically, we are able to visualize both morphology and physico-chemical nature of the bacterial cell surface change in response to copper treatment, leading to the membrane damage and cytoplasm leakage. Overall, the time-lapse AFM imaging combined with hydrophobicity mapping approach presented here provides spatio-temporal insight into the antimicrobial mechanisms of copper at the single cell level, and can be applied to design of better metallic antimicrobial materials as well as investigate different microorganisms.

The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).

Periodical cicadas have proven useful in testing a variety of ecological and evolutionary hypotheses because of their unusual life history, extraordinary abundance, and wide geographical range. Periodical cicadas provide the best examples of synchronous periodicity and predator satiation in the animal kingdom, and are excellent illustrations of habitat partitioning (by the three morphologically distinct species groups), incipient species (the year classes or broods), and cryptic species (a newly discovered 13-year species, Magicicada neotredecim). They are particularly useful for exploring questions regarding speciation via temporal isolation, or allochronic speciation. Recently, data were presented that provided strong support for an instance of allochronic speciation by life-cycle switching. This speciation event resulted in the formation of a new 13-year species from a 17-year species and led to secondary contact between two formerly separated lineages, one represented by the new 13-year cicadas (and their 17-year ancestors), and the other represented by the pre-existing 13-year cicadas. Allozyme frequency data, mitochondrial DNA (mtDNA), and abdominal colour were shown to be correlated genetic markers supporting the life-cycle switching/allochronic speciation hypothesis. In addition, a striking pattern of reproductive character displacement in male call pitch and female pitch preference between the two 13-year species was discovered. In this paper we report a strong association between calling song pitch and mtDNA haplotype for 101 individuals from a single locality within the M. tredecim/M. neotredecim contact zone and a strong association between abdomen colour and mtDNA haplotype. We conclude by reviewing proposed mechanisms for allochronic speciation and reproductive character displacement.

Little is known about the fluids or the microbial communities present within potentially vast hydrothermal reservoirs contained in still-hot volcanic ocean crust beneath the flanks of the mid-ocean ridge. During Alvin dives in 2002, organic material attached to basalt was collected at low, near-ambient temperatures from an abyssal hill fault scarp in 0.5 Ma lithosphere on the western ridge flank of the East Pacific Rise. Mineral analysis by X-ray diffractometry and scanning electron microscopy revealed high-temperature (> 110 degrees C) phases chalcopyrite (Cu(5)FeS(4)) and 1C pyrrhotite (Fe(1-x)S) within the fault scarp materials. A molecular survey of archaeal genes encoding 16S rRNA identified a diverse hyperthermophilic community, including groups within Crenarchaeota, Euryarchaeota, and Korarchaeota. We propose that the sulfide, metals and archaeal communities originated within a basalt-hosted subseafloor hydrothermal habitat beneath the East Pacific Rise ridge flank and were transported to the seafloor during a recent episode of hydrothermal venting from the abyssal hill fault. Additionally, inferred metabolisms from the fault scarp community suggest that an ecologically unique high-temperature archaeal biosphere may thrive beneath the young East Pacific Rise ridge flank and that abyssal hill fault scarps may present new opportunities for sampling for this largely unexplored microbial habitat.

Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food-related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterize their surface mechanically. The progression in elasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.

Microbial biomarkers that indicate aspects of an organism's growth conditions are important targets of forensic research. In this study, we examined fatty acid composition as a signature for the types of complex nutrients in the culturing medium. Bacillus cereus T-strain spores were grown in medium formulations supplemented with one of the following: peptone (meat protein), tryptone (casein protein), soy protein, and brain-heart infusion. Cellular biomass was profiled with fatty acid methyl ester (FAME) analysis. Results showed peptone cultures produced spores enriched in straight-chained lipids. Tryptone cultures produced spores enriched in branched-odd lipids when compared with peptone, soy, and brain-heart formulations. The observed FAME variation was used to construct a set of discriminant functions that could help identify the nutrients in a culturing recipe for an unknown spore sample. Blinded classification tests were most successful for spores grown on media containing peptone and tryptone, showing 88% and 100% correct identification, respectively.

‘Touch’ or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures. This dataset comprises the results from antibody hybridization surveys using Human Leukocyte Antigen (HLA) and Cytokeratin (CK) probes, as well as surveys of optical properties of deposited cells, including forward scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel. All analyses were performed on “touch” samples deposited by several different contributors on multiple days to assess inter- and intra-contributor variability.