A part-declared blind testing program in firearms examination.

In 2015 and 2016 the Central Unit of the Dutch National Police created and submitted 21 cartridge case comparison tests as real cases to the Netherlands Forensic Institute (NFI), under supervision of the University of Twente (UT). A total of 53 conclusions were drawn in these 21 tests. For 31 conclusions the underlying ground truth was "positive", in the sense that it addressed a cluster of cartridge cases that was fired from the same firearm. For 22 conclusions the ground truth was "negative", in the sense that the cartridge cases were fired from different firearms. In none of the conclusions, resulting from examinations under casework conditions, misleading evidence was reported. All conclusions supported the hypothesis reflecting the ground truth. This article discusses the design and results of the tests in more detail.

Finding the way forward for forensic science in the US-A commentary on the PCAST report.

A recent report by the US President's Council of Advisors on Science and Technology (PCAST), (2016) has made a number of recommendations for the future development of forensic science. Whereas we all agree that there is much need for change, we find that the PCAST report recommendations are founded on serious misunderstandings. We explain the traditional forensic paradigms of match and identification and the more recent foundation of the logical approach to evidence evaluation. This forms the groundwork for exposing many sources of confusion in the PCAST report. We explain how the notion of treating the scientist as a black box and the assignment of evidential weight through error rates is overly restrictive and misconceived. Our own view sees inferential logic, the development of calibrated knowledge and understanding of scientists as the core of the advance of the profession.

Implementing context information management in forensic casework: Minimizing contextual bias in firearms examination.

Managing context information in forensic casework aims to minimize task-irrelevant information while maximizing the task-relevant information that reaches the examiner. A design and implementation of context information management (CIM) is described for forensic firearms examination. Guided by a taxonomy of different sources of context information, a flow-chart was constructed that specifies the process of casework examination and context information management. Due to the risk of bias, another examiner may need to be involved when context information management is unsuccessful. Application of context information management does not make a subjective examination objective, but can limit the risks of bias with a minimal investment of time and resources.

Design and results of an exploratory double blind testing program in firearms examination.

In 2010, the Netherlands Forensic Institute (NFI) and the University of Amsterdam (UvA) started a series of tests for the NFI's Firearms Section. Ten cartridge case and bullet comparison tests were submitted by various external parties as regular cases and mixed in the flow of real cases. The results of the tests were evaluated with the VU University Amsterdam (VUA). A total of twenty-nine conclusions were drawn in the ten tests. For nineteen conclusions the submitted cartridge cases or bullets were either fired from the questioned firearm or from one and the same firearm, in tests where no firearm was submitted. For ten conclusions the submitted cartridge cases or bullets were either fired from another firearm than the submitted one or from several firearms, in tests where no firearm was submitted. In none of the conclusions misleading evidence was reported, in the sense that all conclusions supported the true hypothesis. This article discusses the design considerations of the program, contains details of the tests, and describes the various ways the test results were and could be analyzed.

Likelihood ratio methods for forensic comparison of evaporated gasoline residues.

In the investigation of arson, evidence connecting a suspect to the fire scene may be obtained by comparing the composition of ignitable liquid residues found at the crime scene to ignitable liquids found in possession of the suspect. Interpreting the result of such a comparison is hampered by processes at the crime scene that result in evaporation, matrix interference, and microbial degradation of the ignitable liquid. Most commonly, gasoline is used as a fire accelerant in arson. In the current scientific literature on gasoline comparison, classification studies are reported for unevaporated and evaporated gasoline residues. In these studies the goal is to discriminate between samples of several sources of gasoline, based on a chemical analysis. While in classification studies the focus is on discrimination of gasolines, for forensic purposes a likelihood ratio approach is more relevant. In this work, a first step is made towards the ultimate goal of obtaining numerical values for the strength of evidence for the inference of identity of source in gasoline comparisons. Three likelihood ratio methods are presented for the comparison of evaporated gasoline residues (up to 75% weight loss under laboratory conditions). Two methods based on distance functions and one multivariate method were developed. The performance of the three methods is characterized by rates of misleading evidence, an analysis of the calibration and an information theoretical analysis. The three methods show strong improvement of discrimination as compared with a completely uninformative method. The two distance functions perform better than the multivariate method, in terms of discrimination and rates of misleading evidence.

Simulation of generation-collection experiments with homogeneous kinetics: application to electrochemical investigation of superoxide radical anion generation by osteoclasts on bone.

We report simulations of electrochemical generation-collection experiments in which the generator is a small disc producing a specified time-dependent flux of the analyte and the collector is a large planar electrode which collects the analyte at the mass transport-controlled rate. This geometry corresponds to many experiments in bioelectrochemistry where a relatively large sensor is used to detect the products of a cell's metabolism at low concentration. In particular, our simulations are motivated by attempts to understand our results on the detection of the superoxide radical anion burst generated by osteoclasts (bone-resorbing cells) in response to various stimuli. Superoxide is present at low levels and disproportionates in aqueous media; however, the homogeneous kinetics are included in our simulations and the results show that it is possible to estimate the magnitude of the flux of superoxide produced by the cells and to accurately determine the time-dependence of the flux in response to stimuli such as injection of parathyroid hormone, vitamin D(3) and pertussis toxin. In all these cases, the superoxide anion flux was successfully modeled as uniform across the cell surface with time-dependence of the form j(0)e(-k(d)t) + j(∞). j(∞) is the sustained flux of superoxide and the first-order rate constant k(d) and the magnitude j(0) describe the transient component of the flux. The simulations indicate that for cell-electrode gaps D approximately < √(D/k(d)), where D is the diffusion coefficient, the value of k(d) can be accurately extracted from the time-dependence of the collector current without detailed knowledge of parameters which are hard to measure during the experiment, e.g., the cell radius a and cell-electrode separation d. In the case of parathyroid hormone, the first-order rate constant describing the decay of the transient component was k(d) = 1.8 ± 0.8 × 10(-1) s(-1), but much slower decays were observed in response to pertussis toxin (k(d) = 1.5 ± 0.5 × 10(-2) s(-1)) and vitamin D(3) (k(d) = 1.1 ± 0.5 × 10(-3) s(-1)).

Inference of identity of source using univariate and bivariate methods.

In this study we explore the inference of identity of source using a two-dimensional feature vector. As an example, we study the use of the Bayesian framework for the estimation of the value of evidence of color measurements for identity of source of blue ballpoint pen inks. Univariate as well as bivariate analyses are carried out for color data that was acquired with a flatbed scanner. While this might not be the best method to discriminate inks, we will use it as an example to estimate what the value of the evidence is, however low or high it may be. It is hoped that this exercise is instructional, as a similar approach can readily be applied in other situations.

Dispersions of alkyl-capped silicon nanocrystals in aqueous media: photoluminescence and ageing.

Alkyl-capped silicon nanocrystals can be dispersed in aqueous media by shaking or stirring their solutions in organic solvents (DMSO, ether, THF) with excess water. THF is the most straightforward choice with which to prepare stable aqueous dispersions, because the nanocrystals are very soluble in THF and it is also miscible with water. As little as 0.01% v/v tetrahydrofuran is sufficient. DMSO and ether were the preferred choices for subsequent staining of live cells because THF shows some acute toxicity even when very dilute. The luminescence intensity of the aqueous dispersions is linear in particle concentration and independent of pH over the range 5-9. The sols retain their photoluminescence and are stable against flocculation for at least 6 months.

Influence of high tibial osteotomy on bone marrow edema in the knee.

To determine the influence of high tibial osteotomy on subchondral bone marrow edema in medial osteoarthritis of the varus knee, full leg-length radiographs and magnetic resonance imaging were performed in 20 patients (20 knees) before surgery, 1 year postoperatively, and at a mean of 7 years postoperatively. The extent of bone marrow edema in the medial compartment was quantified with magnetic resonance imaging in two planes using the formula for a prolate ellipsoid as follows: length x width x depth x pi/6. We used the Japanese Orthopaedic Association knee score for clinical evaluation. At the last followup, all knees with valgus alignment (10/10) showed reduced edema. In contrast, bone marrow edema increased or remained unchanged in four of 10 knees with neutral or varus alignment. The percentage of satisfactory results was 100% (10/10) in valgus knees and only 30% (3/10) in neutral or varus knees. Extent of bone marrow edema at the followup correlated with the mechanical axis and knee score. Because of the prognostic value of bone marrow abnormalities in the medial compartment observed on magnetic resonance imaging, early lateral closing wedge osteotomy should be considered in patients with varus malalignment and bone marrow edema even in mild cases of medial osteoarthritis.

Spontaneous osteonecrosis of the knee: biochemical markers of bone turnover and pathohistology.

OBJECTIVE: The aim of this study was to evaluate bone metabolism in patients with spontaneous osteonecrosis (ON) of the medial femoral condyle. METHOD: In 22 consecutive patients, undergoing total knee arthroplasty, biochemical markers of bone metabolism were measured in aspirates from cancellous bone and in samples obtained simultaneously from peripheral blood. Specimens of the medial femoral condyle were available for histologic examination and the lesion size, assessed on radiographs, was compared with the results from bone turnover measurements. Twenty patients with osteoarthritis (OA) of the knee served as a control. Bone-specific alkaline phosphatase (bone ALP), osteocalcin (OC), procollagen type I N-terminal propeptide (PINP), and C-terminal cross-linking telopeptide (ICTP) were studied. RESULTS: Mean serum levels of analytes were not different in patients with ON and OA. The serum concentrations averaged 16.2 vs 13.3 ng/mL (OC), 10.2 vs 12.1 ng/mL (bone ALP), 4.6 vs 4.1 ng/mL (ICTP), and 33.2 vs 40.4 ng/mL (PINP) in patients with ON and OA, respectively. In samples obtained from cancellous bone, mean concentrations of all markers were elevated significantly when compared to serum levels. The mean marker concentrations in samples obtained from cancellous bone were 33.8 vs 43.3 ng/mL (OC), 34.6 vs 37.3 ng/mL (bone ALP), 64.8 vs 36.1 ng/mL (ICTP, P=0.02), and 208.0 vs 176.2 ng/mL (PINP) in patients with ON and OA, respectively. The lesion size was at mean 440.5+/-275.8mm(2) in knees with ON and did not correlate with either serum or bone concentrations of all markers tested (P>0.1). CONCLUSION: The marked elevation of markers in samples obtained from cancellous bone pointed at increased turnover in both diseases when compared to healthy individuals. In line with histologic findings of necrosis of subchondral bone, focal degradation of collagen type I was more pronounced in knees with ON. Mean serum concentrations of all markers, however, were not different from healthy individuals and thus did not provide any useful clue in the diagnosis spontaneous ON.

Scanning electrochemical microscopy at the surface of bone-resorbing osteoclasts: evidence for steady-state disposal and intracellular functional compartmentalization of calcium.

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.

ADP binding induces an asymmetry between the heads of unphosphorylated myosin.

Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.

Elevated levels of lipoprotein(a) in familial bone marrow edema syndrome of the hip.

There is controversy whether bone marrow edema syndrome represents a distinct transient disease or reflects an early reversible phase of spontaneous osteonecrosis of the hip. Hypofibrinolysis on the basis of elevated plasma levels of plasminogen activator inhibitor or lipoprotein(a) or both has been reported to favor the development of bone marrow edema syndrome and nontraumatic avascular necrosis. The current authors report on the familial occurrence of transient bone marrow edema syndrome of the hip in three female family members. Plasma concentrations of lipoprotein(a) were elevated in all three patients, whereas serum levels of plasminogen activator inhibitor were within normal ranges. This first familial description of transient bone marrow edema syndrome of the hip strongly suggests that a genetically determined elevation of lipoprotein(a) may be an important predisposing factor in these patients.

Forskolin has a bimodal cAMP-independent effect on superoxide anion generation in isolated osteoclasts.

The mode of action of forskolin is of clinical and scientific interest since forskolin has been shown to have potentially therapeutic bone anti-resorptive and anti-hypertensive properties. Forskolin is thought to inhibit the bone resorptive activity of osteoclasts by elevating cytosolic cAMP and to mimic as well as augment the anti-resorptive effect of calcitonin (CT). Other studies have found that forskolin has a dose-dependent dual effect in mouse calavaria, stimulating bone resorption at low doses and having an inhibitory effect at high doses. However, the acute effect of forskolin on osteoclast functional modality has never been studied. The present investigation examined the effect of low (1 mM) and high doses (10 mM) of forskolin on superoxide anion (O2-) generation in isolated bone-resorbing rat osteoclasts. Forskolin was found to have a bimodal cAMP-independent effect on O2- generation, being stimulatory at a low dose and having an inhibitory effect at a higher dose. These findings also suggest that CT-induced inhibition of O2- generation in the osteoclasts is likely to be mediated by cAMP-independent pathways, perhaps involving [Ca2+]i modulation.

Mitochondrial DNA deletion associated oxidative stress and severe male osteoporosis.

We have screened the mitochondrial genome of 15 men with symptomatic vertebral fractures (median age 62 years, range 27-72 years) and 17 male control subjects (median age 61 years, range 40-73 years) for the presence of mitochondrial DNA (mtDNA) deletions in peripheral monocyte DNA. Polymerase chain reaction analysis provided evidence of a common age-related (4.9 kb) mtDNA deletion situated between nucleotides 8470 and 13.460 of the genomic sequence in 5 of the 17 controls (29%) and 9 of the 15 patients (60%) investigated. Southern blotting and polymerase chain reaction revealed a novel 3.7 kb deletion in 2 patients. One of the affected patients, a 27-year-old man with severe osteoporosis (lumbar spine bone mineral density (BMD) 0.381 g/cm(2); Z-score -6.45) was found to harbor deletion in almost 50% of the mitochondria. The patient had a blood lactic acid level (4.6 nM) that was over 3 times the upper reference range (0-1.3 mM), thus confirming the presence of systemic oxidative stress. Further analysis by modified primer shift polymerase chain reaction showed the 5' breakpoint to be between the nucleotides 10.63 kb and 10.80 kb of the mtDNA. The second patient harboring the 3.7 kb deletion was older (62 years) with less severe osteoporosis (lumbar spine BMD 0.727/cm(2); Z-score -2.58) and the proportion of affected mitochondria was lower (25%). The significance of these findings is discussed and the possible relation between oxidative stress and accelerated bone loss is examined.

Direct non-genomic effect of steroid hormones on superoxide anion generation in the bone resorbing osteoclasts.

We have investigated the possible acute effect of steroid hormones, including 1alpha,25 dihydroxycholecalciferol (1alpha,25(OH)2D3) and estradiol, on the generation of superoxide anion (O2*-) in bone resorbing osteoclasts. Evidence is presented demonstrating acute non-genomic stimulatory action of 1alpha,25(OH)2D3 on the production of free radicals by rat osteoclasts cultured on calcified matrix. The increase in O2*- production was observed in the range of 6-10 s (n = 5) following exposure of enriched osteoclasts to 1alpha,25(OH)2D3 and was found to be transient with the peak response being in the range of 5-45 s (n = 5). The decline in the transient was much slower than the elevation, time for the decay being in the range 1-5 min (n = 5) and remained above the levels present prior to the addition. The exposure of the osteoclast to dexamethasone was found to have no effect on O2*- generation, whilst estradiol was found to be inhibitory. The mode of stimulation and the kinetics of the transients of O2*- in the bone resorbing osteoclasts produced by 1alpha,25(OH)2D3 were similar to that of parathyroid hormone (PTH) and pertussis. The exposure of the bone resorbing osteoclasts to cholera toxin was found to have no effect, suggesting that the stimulatory action is unlikely to be mediated via cAMP elevation. The importance of these observations is discussed in the context of calcium homeostasis and bone physiology.

cAMP-dependent inhibition is dominant in regulating superoxide production in the bone-resorbing osteoclasts.

Calciotropic hormones such as parathyroid hormone (PTH) and calcitonin have been shown to have stimulatory and inhibitory effects respectively on superoxide anion (O2-) generation by osteoclasts, but the exact intracellular signalling mediating these pathways has not been investigated. In order to elucidate the intracellular pathways controlling O2- generation, we have carried out a systematic study of the effect of different agents on O2- production in osteoclasts cultured on bovine cortical bone. Dibutyryl cAMP and cholera toxin, while having no effect on the basal level of O2- production in bone-resorbing osteoclasts, were, however, found to completely block the stimulation of free radical production by PTH, pertussis toxin and ionomycin. The stimulation of O2- production was found to be independent of protein kinase C-dependent pathways since the presence of bisindolylmaleimide (GF109203X) (1 microM) did not block stimulation by PTH and pertussis toxin. Interestingly, while exposure to bisindolylmaleimide at this concentration did not have any effect on the basal level of O2- production, exposure to a higher concentration (10 microM), which is known to inhibit both protein kinase C and A, produced significant stimulation. These in vitro findings suggest that in the bone-resorbing cells, cAMP-dependent protein kinases prevent further stimulation of NADPH oxidase by agents such as PTH and pertussis toxin. The increase in cAMP has also been recently demonstrated to be associated with down-regulation of the oxidative burst in adherent neutrophils; and the findings reported here suggest a similar role for cAMP in O2- generation in osteoclasts cultured on bone.