Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows. Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1-18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing.

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.

Improvised first aid techniques for terrorist attacks.

Terrorist acts occur every day around the world. Healthcare professionals are often present as bystander survivors in these situations, with none of the equipment or infrastructure they rely on in their day-to-day practice. Within several countries there has been a move to disseminate the actions to take in the event of such attacks: in the UK, Run, Hide, Tell, and in the USA, Fight Back This paper outlines how a very basic medical knowledge combined with everyday high-street items can render highly effective first aid and save lives. We discuss and summarise modern improvised techniques. These include the ABCDE approach of treating catastrophic haemorrhage before airway management, bringing together improvised techniques from the military and wilderness medicine. We explain how improvised tourniquets, wound dressings, splinting and traction devices can be fabricated using items from the high street: nappies, tampons, cling film, duct tape and tablecloths. Cervical spine immobilisation is a labour-intensive protocol that is often practised defensively. With little evidence to support the routine use of triple immobilisation, this should be replaced with a common sense dynamic approach such as the Montana neck brace. Acid or alkali attacks are also examined with simple pragmatic advice. Analgesia is discussed in the context of a prehospital setting. Pharmacy-obtained oral morphine and diclofenac suppositories can be used to treat moderate pain without relying on equipment for intravenous/intraosseous infusion in prolonged hold situations. The differentiation between concealment and cover is summarised: scene safety remains paramount.

Combat Trousers as Effective Improvised Pelvic Binders A Comparative Cadaveric Study.

BACKGROUND: Improvised explosive devices and landmines can cause pelvic fractures, which, in turn, can produce catastrophic hemorrhage. This cadaveric study compared the intrapelvic pressure changes that occurred with the application of an improvised pelvic binder adapted from the combat trousers worn by British military personnel with the commercially available trauma pelvic orthotic device (TPOD). METHODS: Six unembalmed cadavers (three male, three female) were used to simulate an unstable pelvic fracture with complete disruption of the posterior arch (AO/OTA 61-C1) by dividing the pelvic ring anteriorly and posteriorly. A 3-4cm manometric balloon filled with water was placed in the retropubic space and connected to a 50mL syringe and water manometer via a three-way tap. A baseline pressure of 8cm H2O (average central venous pressure) was set. The combat trouser binder (CTB) and TPOD were applied to each cadaver in a random sequence and the steady intrapelvic pressure changes were recorded. Statistical analysis was performed using the Wilcoxon rank-sum test and a paired t test depending on the normality of the data to determine impact on the intrapelvic pressure of each intervention compared with baseline. RESULTS: The median steady intrapelvic pressure achieved after application of the CTB was 16cm H2O and after application of the TPOD binder was 18cm H2O, both of which were significantly greater than the baseline pressure (ρ < .01 and .036, respectively) but not significantly different from each other (ρ > .05). CONCLUSION: Pelvic injuries are increasingly common in modern theaters of war. The CTB is a novel, rapidly deployable, yet effective, method of pelvic binding adapted from the clothes the casualty is already wearing. This technique may be used in austere environments to tamponade and control intrapelvic hemorrhage.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Intra-pelvic pressure changes after pelvic fracture: A cadaveric study quantifying the effect of a pelvic binder and limb bandaging over a bolster.

INTRODUCTION: Unstable pelvic fractures can be life-threatening due to catastrophic haemorrhage. Non-invasive methods of reducing and stabilising these injuries include pelvic binder application and also lower limb bandaging over a knee-flexion bolster. Both of these methods help close the pelvic ring and should tamponade bleeding. This study aimed to quantify the intra-pelvic pressure changes that occurred with 3 different manoeuvres: lower limb bandaging over a bolster; a Trauma Pelvic Orthotic Device (T-POD) pelvic binder, and a combination of both. METHODS: Following a pilot study with 2 soft embalmed cadavers, a formal study with 6 unembalmed cadavers was performed. For each specimen an unstable pelvic injury was created (OA/OTA 61-C1) by dividing the pelvic ring anteriorly and posteriorly. A 3-4cm manometric water-filled balloon was placed in the retropubic space and connected to a 50ml syringe and water manometer via a 3-way tap. A baseline pressure of 8cmH2O (equating to the average central venous pressure) was used for each cadaver. Steady intra-pelvic pressures (more reliably reflecting the pressures achieved following an intervention) were used in the subsequent statistical analysis, using R statistical language and Rstudio. Paired t-test or Wilcoxon's rank sum test were used (depending on the normality of the dataset) to determine the impact of each intervention on the intra-pelvic pressure. RESULTS: The mean steady intra-pelvic pressures were significantly greater than the baseline pressure for each intervention. The binder and limb bandaging over a bolster alone increased the mean steady pelvic pressures significantly to 24 (SE=5) (p<0.036) and 15.5 (SE=2) (p<0.02)cmH2O respectively. Combining these interventions further increased the mean steady pressure to 31 (SE=7)cmH2O. However, this was not significantly greater than pressures for each of the individual interventions. DISCUSSION: Both lower limb bandaging over a bolster and pelvic binder application significantly increased intra-pelvic pressure above the baseline pressure. This was further increased through combining these interventions, which could be useful clinically to augment haemorrhage control in these fractures. CONCLUSION: Lower-limb bandaging over a bolster, and pelvic binder application, both significantly increased intra-pelvic pressures, and were greatest in combination. These findings support the use of these techniques to facilitate non-surgical haemorrhage control.

Light Sheet Fluorescence Microscopy (LSFM).

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.

Robust measurement of membrane bending moduli using light sheet fluorescence imaging of vesicle fluctuations.

The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons between disparate techniques applied to the same systems. We introduce here the use of selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) to image thermal fluctuations of giant vesicles. The optical sectioning of SPIM enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For both lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, which lowers membrane rigidity in a concentration-dependent manner, we show that the resulting bending modulus values are in close agreement with those derived from an independent assay based on membrane tether pulling. We also show that the fluctuation spectra of vesicles bound by the mammalian Sar1A protein, which stiffens membranes at high concentrations, are not well fit by a model of homogeneous quasi-spherical vesicles, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties induced by protein assemblies.

Modulation of membrane rigidity by the human vesicle trafficking proteins Sar1A and Sar1B.

The sculpting of membranes into highly curved vesicles is central to intracellular cargo trafficking, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical trap based assay that measures in vitro membrane response to imposed deformations, we examined the behavior of the two human paralogs of Sar1, a key component of the COPII family of vesicle coat proteins. Like their yeast counterpart, the human Sar1 proteins can lower the mechanical rigidity of the membranes to which they bind. Unlike the yeast Sar1, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which protein clustering governs membrane mechanical properties.

The vesicle trafficking protein Sar1 lowers lipid membrane rigidity.

The sculpting of membranes into dynamic, curved shapes is central to intracellular cargo trafficking. Though the generation of membrane curvature during trafficking necessarily involves both lipids and membrane-associated proteins, current mechanistic views focus primarily on the formation of rigid cages and curved scaffolds by protein assemblies. Here we report on a different mechanism for the control of membrane deformation, unrelated to the imposition of predefined curvature, involving modulation of membrane material properties: Sar1, a GTPase that regulates vesicle trafficking from the endoplasmic reticulum, lowers the rigidity of the lipid bilayer membrane to which it binds. In vitro assays in which optically trapped microspheres create controlled membrane deformations revealed a monotonic decline in bending modulus as a function of Sar1 concentration, down to nearly zero rigidity, indicating a dramatic lowering of the energetic cost of curvature generation. This is the first demonstration that a vesicle trafficking protein lowers the rigidity of its target membrane, leading to a new conceptual framework for vesicle biogenesis.

A thin film detection/response system for pathogenic bacteria.

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5alpha. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle-bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli.

Monolayer-protected nanoparticle film assemblies as platforms for controlling interfacial and adsorption properties in protein monolayer electrochemistry.

Assembled films of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), are investigated as adsorption platforms in protein monolayer electrochemistry (PME), a strategy for studying the electron transfer (ET) of redox proteins. Modified electrodes featuring MPC films assembled with various linking methods, including both electrostatic and covalent mechanisms, are employed to immobilize cytochrome c (cyt c) for electrochemical analysis. The background signal (non-Faradaic current) of these systems is directly related to the structure and composition of the MPC films, including nanoparticle core size, protecting ligand properties, as well as the linking mechanism utilized during assembly. Dithiol-linked films of Au225(C6)75 are identified as optimal films for PME by sufficiently discriminating against detrimental background current and exhibiting interfacial properties that are readily engineered for cyt c adsorption and electroactivity (Faradaic current). Surface concentrations and denaturation rates of adsorbed cyt c are dictated by specific manipulation of the individual MPCs composing the outer layer of the film. The use of specially designed, hydrophilic MPCs as a terminal film layer results in near-ideal cyt c voltammetry, attributed to a high degree of molecular level control of the necessary interfacial interactions and flexibility needed to create a uniform and effective binding of protein across large areas of a substrate. The electrochemical properties of cyt c at MPC films, including ET rate constants that are unaffected by the large ET distance introduced by MPC assemblies, are compared to traditional strategies employing self-assembled monolayers to immobilize cyt c. The incorporation of nanoparticles as protein adsorption platforms has implications for biosensor engineering as well as fundamental biological ET studies.

Covalently networked monolayer-protected nanoparticle films.

Covalently networked films of nanoparticles can be assembled on various substrates from functionalized monolayer-protected clusters (MPCs) via ester coupling reactions. Exposure of a specifically modified substrate to alternating solutions of 11-mercaptoundecanoic acid exchanged and 11-mercaptoundecanol exchanged MPCs, in the presence of ester coupling reagents, 1,3-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, results in the formation of a multilayer film with ester bridges between individual nanoparticles. These films can be grown in a controlled manner to various thicknesses and exhibit certain properties that are consistent with films having other types of interparticle connectivity, including chemical vapor response behavior and quantized double layer charging. Ester coupling of MPCs into assembled films is a straightforward and highly versatile approach that results in robust films that can endure harsher chemical environments than other types of films. The stability of these covalent films is assessed and compared to other more traditional MPC film assemblies.