Individualised and non-contact post-mortem interval determination of human bodies using visible and thermal 3D imaging.

Determining the time since death, i.e., post-mortem interval (PMI), often plays a key role in forensic investigations. The current standard PMI-estimation method empirically correlates rectal temperatures and PMIs, frequently necessitating subjective correction factors. To overcome this, we previously developed a thermodynamic finite-difference (TFD) algorithm, providing a rigorous method to simulate post-mortem temperatures of bodies assuming a straight posture. However, in forensic practice, bodies are often found in non-straight postures, potentially limiting applicability of this algorithm in these cases. Here, we develop an individualised approach, enabling PMI reconstruction for bodies in arbitrary postures, by combining photogrammetry and TFD modelling. Utilising thermal photogrammetry, this approach also represents the first non-contact method for PMI reconstruction. The performed lab and crime scene validations reveal PMI reconstruction accuracies of 0.26 h ± 1.38 h for true PMIs between 2 h and 35 h and total procedural durations of ~15 min. Together, these findings broaden the potential applicability of TFD-based PMI reconstruction.

Quantitative aortography for assessing aortic regurgitation after transcatheter aortic valve implantation: results of the multicentre ASSESS-REGURGE Registry.

AIMS: Quantitative aortography using videodensitometry is a valuable tool for quantifying paravalvular regurgitation after TAVI, especially in the minimalist approach - without general anaesthesia. However, retrospective assessment of aortograms showed moderate feasibility of assessment. We sought to determine the prospective feasibility of quantitative aortography after a protocol of acquisition. METHODS AND RESULTS: This was a multicentre registry in Japan, Canada, the Netherlands and Germany including consecutive patients with Heart Team indication to undergo TAVI over a median period of 12 months. Operators performed final aortograms according to a pre-planned projection (either by CT or visually - Teng's rule). An independent core laboratory (Cardialysis) analysed all images for feasibility and for regurgitation assessment. From the four centres included in the present analysis, a total of 354 patients underwent TAVI following the acquisition protocol and all the aortograms were analysed by the core lab. The analyses were feasible in 95.5% (95% confidence interval [CI]: 93.2% to 97.5%) of the cases. This rate of analysable assessment was significantly higher than the feasibility in previous validation studies, such as in the RESPOND population (95.5% vs. 57.5%, p<0.0001). No differences were observed among different planning strategies (CT 96.5% vs. Teng's rule 93%, p=0.159; or Circle 98.5% vs. 3mensio 95.8% vs. Teng's rule 93%, p=0.247). CONCLUSIONS: ASSESS-REGURGE showed a high feasibility of assessment of regurgitation with quantitative aortography with protocoled acquisition. This may be of great importance for quantifying regurgitation in TAVI procedures (optimisation, guidance of post-dilatation), and in future clinical trials, in order to address sealing features of novel devices for TAVI objectively. ClinicalTrials.gov Identifier: NCT03644784.

Tracers as invisible evidence - The transfer and persistence of flock fibres during a car exchange.

This study assessed the recovery of flock fibres used as a tracer in a car exchange scenario. Flock fibres were deposited onto a car seat (or model thereof) and their transfer and persistence was investigated after a real or simulated car exchange. The overall aim of this study was to achieve an optimal use of flock fibres as tracers, i.e. to be able to select a fit-for-purpose flock fibre, to be able to predict the amount of flock fibres to be recovered from crime related items, and to be able to use these numbers to exclude accidental uptake. The effect of a number of variables on the transfer and persistence of flock fibres was studied, including flock fibre length, car upholstery, and trousers material. Laboratory based experiments were undertaken first, followed by realistic field based experiments. The flock fibres were captured in a non-destructive manner through fluorescence photography. A Matlab algorithm enabled fast automated counting of flock fibres on the images. Results indicate that an initial rapid loss of flock fibres from garments may be expected as a result of moderate movement. Although the amount of flock fibres to be recovered is affected by the flock fibre length, the type of car upholstery, and the type of trousers materials (if frictional force is taken into consideration), large numbers of flock fibres have been recovered from all target materials throughout the transfer route. These numbers are higher than the amount of flock fibres recovered due to accidental uptake. In conclusion, flock fibres can serve as invisible evidence to reconstruct a series of events.

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy.

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Infrared imaging of the crime scene: possibilities and pitfalls.

All objects radiate infrared energy invisible to the human eye, which can be imaged by infrared cameras, visualizing differences in temperature and/or emissivity of objects. Infrared imaging is an emerging technique for forensic investigators. The rapid, nondestructive, and noncontact features of infrared imaging indicate its suitability for many forensic applications, ranging from the estimation of time of death to the detection of blood stains on dark backgrounds. This paper provides an overview of the principles and instrumentation involved in infrared imaging. Difficulties concerning the image interpretation due to different radiation sources and different emissivity values within a scene are addressed. Finally, reported forensic applications are reviewed and supported by practical illustrations. When introduced in forensic casework, infrared imaging can help investigators to detect, to visualize, and to identify useful evidence nondestructively.

HDR monotherapy for prostate cancer: a simulation study to determine the effect of catheter displacement on target coverage and normal tissue irradiation.

PURPOSE: The aim of this study was to systematically analyse the effect of catheter displacements both on target coverage and normal tissue irradiation in fractionated high dose rate (HDR) prostate brachytherapy, using a simulation study, and to define tolerances for catheter displacement ensuring that both target coverage and normal tissue doses remain clinically acceptable. Besides the effect of total implant displacement, also displacements of catheters belonging to selected template rows only were evaluated in terms of target coverage and normal tissue dose, in order to analyse the change in dose distribution as a function of catheter dwell weight and catheter location. MATERIAL AND METHODS: Five representative implant geometries, with 17 catheters each, were selected. The clinical treatment plan was compared to treatment plans in which an entire implant displacement in caudal direction over 3, 5, 7 and 10 mm was simulated. Besides, treatment plans were simulated considering a displacement of either the central, most ventral or most dorsal catheter rows only, over 5 mm caudally. RESULTS: Due to displacement of the entire implant the target coverage drops below the tolerance of 93% for all displacements studied. The effect of displacement of the entire implant on organs at risk strongly depended on the patient anatomy; e.g., for 80% of the implant geometries the V(80) of the rectum exceeded its tolerance for all displacements. The effect of displacement of catheters belonging to selected template rows depended strongly on the relative weight of each catheter row when considering the target coverage and on its location when considering the dose in the organs at risk. CONCLUSION: This study supports the need for a check of the catheter locations before each fraction and correction of deviations of the catheter position exceeding 3mm.

Characterization of a daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus strain in a patient with endocarditis.

We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.

Concept for the traceability of fluorescence (beads) in flow cytometry: exploiting saturation and microscopic single molecule bleaching.

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.

Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells.

BACKGROUND/AIMS: This study investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors. METHODS: Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus, E. coli cytosine deaminase (BCDase) suicide fusion proteins were generated. RESULTS: TLM fusion proteins formed hexamers (as do parental wtBCDase proteins) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtBCDase proteins. However, only BCDase-TLM fusion proteins, but not TLM-BCDase fusion nor parental wtBCDase proteins were found to be taken up to the cytoplasm of target cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of BCDase-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since BCDase-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-FC strongly is suggested. CONCLUSIONS: Future therapeutic local application of cell-permeable BCDase-TLM fusion proteins together with a systemic 5-FC prodrug application could result in profound antitumor activities without apparent side effects.

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle.

Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.