Forensically relevant anatomical brain regions cannot be sub-differentiated by RNA expression analysis.

The contextualization of biological traces generated by severe head injuries can be beneficial for criminal investigations. Here we aimed to identify and validate mRNA candidates for a robust sub-differentiation of forensically and traumatologically relevant brain regions. To this purpose, massively parallel sequencing of whole transcriptomes in sample material taken from four different areas of the cerebral cortex (frontal, temporal, parietal, occipital lobe) was performed, followed by bioinformatical data analysis, classification, and biostatistical candidate selection. Candidates were evaluated by Multiplex-RT-PCR and capillary electrophoresis. Only a weak relative upregulation and solely for candidates expressed in the parietal lobe was observed. Two candidates with upregulation in the cerebellar region (PVALB and CDR2L) were chosen for further investigation; however, PVALB could not reliably and repeatedly be detected in any lobe whereas CDR2L was detectable in all lobes. Consequently, we suggest that differences in mRNA expression between four regions of the cerebral cortex are too small and less pronounced to be useful for and applicable in forensic RNA analysis. We conclude that sub-differentiation of these brain regions via RNA expression analysis is generally not feasible within a forensic scope.

Ten years of molecular ballistics-a review and a field guide.

Molecular ballistics combines molecular biological, forensic ballistic, and wound ballistic insights and approaches in the description, collection, objective investigation, and contextualization of the complex patterns of biological evidence that are generated by gunshots at biological targets. Setting out in 2010 with two seminal publications proving the principle that DNA from backspatter collected from inside surfaces of firearms can be retreived and successfully be analyzed, molecular ballistics covered a lot of ground until today. In this review, 10 years later, we begin with a comprehensive description and brief history of the field and lay out its intersections with other forensic disciplines like wound ballistics, forensic molecular biology, blood pattern analysis, and crime scene investigation. In an application guide section, we aim to raise consciousness to backspatter traces and the inside surfaces of firearms as sources of forensic evidence. Covering crime scene practical as well as forensic genetic aspects, we introduce operational requirements and lay out possible procedures, including forensic RNA analysis, when searching for, collecting, analyzing, and contextualizing such trace material. We discuss the intricacies and rationales of ballistic model building, employing different tissue, skin, and bone simulants and the advantages of the "triple-contrast" method in molecular ballistics and give advice on how to stage experimental shootings in molecular ballistic research. Finally, we take a look at future applications and prospects of molecular ballistics.

Nothing but hot air?-On the molecular ballistic analysis of backspatter generated by and the hazard potential of blank guns.

Blank cartridge guns are prevalent especially in countries with laws restricting access to conventional firearms, and it is a common misconception that these weapons are harmless and only used as toys or for intimidation. However, although their harming potential is well-documented by numerous reports of accidents, suicides, and homicides, a systematic molecular biological investigation of traces generated by shots from blank cartridges at biological targets has not been done so far. Herein, we investigate the occurrence and analyzability of backspatter generated by shots of different types of blank cartridge guns firing different types of blank ammunition at ballistic gelatin model cubes doped with human blood and radiological contrast agent soaked into a spongious matrix and covered with three different variants of skin simulants. All skin simulants were penetrated, and backspatter was created in 100% of the shots in amounts sufficient for forensic short tandem repeat (STR) typing that resulted in the correct identification of the respective blood donor. Visible backspatter was documented on the muzzle and/or inside the barrel in all cases, and in 75% of cases also on the outer surfaces and on the shooter's hand(s). Wound cavities were measured and ranged between 1 and 4.5 cm in depth. Discussing our findings, we provide recommendations for finding, recovering, and analyzing trace material from blank guns, and we demonstrate the considerable hazard potential of these devices, which is further emphasized by the presentation of a comprehensive overview of the pertinent literature on injuries inflicted by blank guns.

A distant relationship?-investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance.

In molecular ballistics, where traces originating from the use of firearms against biological targets are investigated, "backspatter" traces are of particular importance. This biological material comprising blood and tissue from the victim is propelled back from the bullet entry site towards the direction of the shooter and can consolidate and persist on the inner and outer surfaces of the firearm, from where it can be collected and analyzed. Thus, a connection between the weapon and the victim can be established solely by molecular biological trace analysis. For the criminalistic investigation of gun-related crimes, the determination of the distance between the weapon and the victim can be of critical importance in reconstructing the circumstances of a crime. In this study, we investigated possible correlations between the shooting distance and the amount of backspatter in/on the used firearm. To this purpose, we employed a previously established skull model and performed shootings in triplicates from various distances up to 50 cm with two types of handguns (pistol and revolver). Backspatter was collected from various sampling locations, and DNA contents were quantified. A post-shooting wound channel evaluation was conducted by optical and radiological evaluation. The obtained DNA yields varied considerably between replicates from the same and from different distances. In contrast, apart from contact shots, no meaningful differences were observable in wound channel evaluations. In summary, no meaningful correlation between backspatter distribution and DNA yields, the shooting distance and the condition of the wound channel could be established.

A Musashi Splice Variant and Its Interaction Partners Influence Temperature Acclimation in Chlamydomonas.

Microalgae contribute significantly to carbon fixation on Earth. Global warming influences their physiology and growth rates. To understand algal short-term acclimation and adaptation to changes in ambient temperature, it is essential to identify and characterize the molecular components that sense small temperature changes as well as the downstream signaling networks and physiological responses. Here, we used the green biflagellate alga Chlamydomonas reinhardtii as a model system in which to study responses to temperature. We report that an RNA recognition motif (RRM)-containing RNA-binding protein, Musashi, occurs in 25 putative splice variants. These variants bear one, two, and three RRM domains or even lack RRM domains. The most abundant Musashi variant, 12, with a molecular mass of 60 kD, interacts with two clock-relevant members of RNA metabolism, the subunit C3 of the RNA-binding protein CHLAMY1 and the 5'-3' exoribonuclease XRN1. These proteins are able to integrate temperature information by up- or down-regulation of their protein levels in cells grown at low (18°C) or high (28°C) temperature. We further show that the 60-kD Musashi variants with three RRM domains can bind to (UG)7 repeat-containing RNAs and are up-regulated in cells grown at a higher temperature during early night. Intriguingly, the 60-kD Musashi variant 12, as well as C3 and XRN1, confer thermal acclimation to C. reinhardtii, as shown with mutant lines. Our data suggest that these three proteins of the RNA metabolism machinery are key members of the thermal signaling network in C. reinhardtii.

Evaluation of the backspatter generation and wound profiles of an anatomically correct skull model for molecular ballistics.

Molecular ballistics connects the molecular genetic analysis of biological traces with the wounding events and complex forensic traces investigated in terminal ballistics. Backspatter, which originates from a projectile hitting a biological target when blood and/or tissue is propelled back into the direction of the gun, is of particular interest; those traces can consolidate and persist on the outer and inner surfaces of firearms and serve as evidence in criminal investigations. Herein, we are the first to present an anatomically correct head model for molecular ballistic research based on a polyurethane skull replica enclosing tissue-simulating sponge material that is doped with "triple-contrast" mixture (EDTA-blood, acrylic paint, and an x-ray contrast agent). Ten percent ballistic gelatin was used as brain simulant. We conducted contact and intermediate-range shots with a Glock 19 pistol (9 mm Luger), a pump-action shotgun (12/70 slugs), and blank cartridge handguns. Each shot was documented by a high-speed camera at 35,000 fps. Apart from the blank cartridge guns, all gunshots penetrated the skull model and created backspatter, which was recovered from the distal part of the barrels and analyzed. The pistol contact shots and one of three shotgun shots yielded full STR profiles. While the shotgun slugs destroyed the skulls, the remaining models could be used for radiological and optical fracture and wound channel evaluation. Known backspatter mechanisms and their respective timing could be confirmed visually by video analysis. Our complete model setup proved to be well applicable to molecular ballistic research as well as wound channel and fracture pattern investigation.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models.

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

DNA transfer to firearms in alternative realistic handling scenarios.

Firearms are the most relevant items of evidence in gun-related crimes, likely bearing various traces facilitating an objective reconstruction of the crime. Trace DNA recovered from firearm surfaces might help to identify individual(s) having handled the firearm and thereby possibly to link the firearm and the corresponding shooter, however, the interpretation of DNA traces on handled items can be challenging and requires a detailed understanding of various factors impacting DNA prevalence, transfer, persistence and recovery. Herein, we aimed at improving our understanding of factors affecting the variability of trace DNA characteristics recovered from firearms handled in gun-related crimes: Skin contact traces were recovered from various outer surfaces of two types of firearms handled in four realistic, casework-relevant handling scenarios and the corresponding trace characteristics (DNA yield, number of contributors, relative profile contribution for known and unknown contributors, LRs) were compared. Trace DNA characteristics differed distinctly between handling conditions, firearm and surface types as well as handling individuals and intraindividual deposits emphasizing the variability and complexity of trace DNA profile composition expected to be recovered from firearms after realistic handling scenarios. The obtained results can provide useful insights for forensic experts evaluating alternative activity level propositions in gun-related crimes.