Photogrammetry using visible, infrared, hyperspectral and thermal imaging of crime scenes.

Photogrammetry is a method for obtaining virtual 3D models of objects and scenes. The technique is increasingly used to record the crime scene in its original, undisturbed state for mapping, analytical and reconstruction purposes. Recently, it was shown that it is possible to visualize and/or chemically analyze latent traces by using advanced cameras which either operate in wavelength ranges beyond the visible range, and/or are able to obtain spectrally resolved images. The combination of these advanced cameras and photogrammetric techniques enables the 3D registration of valuable information. We successfully explored the feasibility to obtain visible, infrared, hyperspectral and thermal 3D registrations of simulated crime scenes using photogrammetry, and demonstrate the possibilities and practical challenges for use in forensic practice.

Visual characteristics for sequencing of overlapping bloodstain patterns.

The deposition sequence of overlapping bloodstain patterns can be valuable reconstructive information. A formal method for sequencing overlapping bloodstain patterns has yet to be published. We present a method for sequencing overlapping transfer and drip stains using visual characteristics. A survey was held amongst educated bloodstain pattern analysts to determine whether the newly acquired method will assist them in correctly sequencing these overlapping bloodstain patterns. Results showed a significant improvement of expert decisions: the percentage of overlapping stains correctly sequenced by participants increased from 57 to 79% using the visual characteristics defined in this study. These results suggest that a decision support system can be built, which helps investigators at the crime scene to sequence overlapping bloodstain patterns.

Hyperspectral imaging for non-contact analysis of forensic traces.

Hyperspectral imaging (HSI) integrates conventional imaging and spectroscopy, to obtain both spatial and spectral information from a specimen. This technique enables investigators to analyze the chemical composition of traces and simultaneously visualize their spatial distribution. HSI offers significant potential for the detection, visualization, identification and age estimation of forensic traces. The rapid, non-destructive and non-contact features of HSI mark its suitability as an analytical tool for forensic science. This paper provides an overview of the principles, instrumentation and analytical techniques involved in hyperspectral imaging. We describe recent advances in HSI technology motivating forensic science applications, e.g. the development of portable and fast image acquisition systems. Reported forensic science applications are reviewed. Challenges are addressed, such as the analysis of traces on backgrounds encountered in casework, concluded by a summary of possible future applications.

Differential translation and fragile X syndrome.

Fragile X syndrome (FXS) is caused by the transcriptional silencing of the Fmr1 gene, which encodes a protein (FMRP) that can act as a translational suppressor in dendrites, and is characterized by a preponderance of abnormally long, thin and tortuous dendritic spines. According to a current theory of FXS, the loss of FMRP expression leads to an exaggeration of translation responses linked to group I metabotropic glutamate receptors. Such responses are involved in the consolidation of a form of long-term depression that is enhanced in Fmr1 knockout mice and in the elongation of dendritic spines, resembling synaptic phenotypes over-represented in fragile X brain. These observations place fragile X research at the heart of a long-standing issue in neuroscience. The consolidation of memory, and several distinct forms of synaptic plasticity considered to be substrates of memory, requires mRNA translation and is associated with changes in spine morphology. A recent convergence of research on FXS and on the involvement of translation in various forms of synaptic plasticity has been very informative on this issue and on mechanisms underlying FXS. Evidence suggests a general relationship in which the receptors that induce distinct forms of efficacy change differentially regulate translation to produce unique spine shapes involved in their consolidation. We discuss several potential mechanisms for differential translation and the notion that FXS represents an exaggeration of one 'channel' in a set of translation-dependent consolidation responses.

Theoretical neuroanatomy and the connectivity of the cerebral cortex.

Over recent years, a wealth of neuroanatomical information on the pattern of interconnections between segregated areas of the cerebral cortex has become available. Here, we describe a set of structural measures, based on graph theory, which can be used to analyze these anatomical patterns. We describe relationships between these structural measures and measures based on patterns of functional connectivity, i.e. patterns of correlations in neural activity. We find that networks capable of producing highly complex functional dynamics share common structural motifs. These motifs are also found in cortical connection matrices, which are characterized by the existence of densely linked groups of areas, low potential wiring length, and a high abundance of reciprocal connections and short cycles. An analysis of cortical functional connectivity demonstrates the existence of functional clusters of highly interactive areas, producing highly complex dynamics. The combined structural and functional analysis outlined in this chapter provides insight into the large-scale functional organization of distributed cortical systems.

Efficient marking of neural stem cell-derived neurons with a modified murine embryonic stem cell virus, MESV2.

Treatments for nervous system disorders that involve transplanting genetically modified neural stem cells may ultimately be feasible. As a step towards this therapeutic approach, a novel murine embryonic stem cell gammaretroviral vector was developed with features designed to optimize transgene expression in neural stem cells and to increase vector safety. All potential start sites of translation in the 5' leader were removed. These sites may compete with an inserted transgene for translation initiation, and also produce potentially immunogenic peptides. Further, all of the gag gene sequences were replaced with a well-defined constitutive transport element from avian leukemia virus to promote nuclear export of viral RNA, and to eliminate any homology between the vector and a murine leukemia virus-derived gag-pol packaging plasmid. Two versions of the virus were made in which EGFP expression was driven either by the Rous sarcoma virus U3 enhancer or by a combination of sequences from the Syn1 and Pgk-1 promoters. Both of these viruses efficiently transduced neural stem cells isolated from embryonic rat hippocampus, and robust EGFP expression was observed in neurons derived from these cells following differentiation in vitro.

Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein.

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.

Degeneracy and complexity in biological systems.

Degeneracy, the ability of elements that are structurally different to perform the same function or yield the same output, is a well known characteristic of the genetic code and immune systems. Here, we point out that degeneracy is a ubiquitous biological property and argue that it is a feature of complexity at genetic, cellular, system, and population levels. Furthermore, it is both necessary for, and an inevitable outcome of, natural selection.

Demonstrations of a generation effect in context memory.

Generation often leads to increased memorability within a laboratory context (see, e.g., Slamecka & Graf, 1978). Of interest in the present study is whether the benefits of generation extend beyond item memory to context memory. To investigate this question, in three experiments, we asked subjects to remember in which of two contexts they had read or generated words. In Experiment 1, the contexts were two different rooms; in Experiment 2A, the contexts were two different computer screens; in Experiment 2B, the contexts were different perceptual characteristics of the to-be-remembered words. In all experiments, subjects were better at remembering the context of generated words than of read words.

Solution structure of the third immunoglobulin domain of the neural cell adhesion molecule N-CAM: can solution studies define the mechanism of homophilic binding?

Homophilic binding of the neural cell adhesion molecule (N-CAM) mediates the calcium-independent cell-cell adhesion that is involved in neuronal development. Two hypotheses have been advanced for the mechanism of homophilic binding. Cell-based experiments have implicated each of the five extracellular immunoglobulin (Ig) domains of N-CAM in the homophilic adhesion interaction, and have predicted that the third domain (Ig III) self-associates. The alternative hypothesis is based on solution observations, which implicate a specific antiparallel interaction between the first two Ig domains (Ig I and Ig II). In order to test these hypotheses, we have determined a high-resolution solution structure of recombinant Ig III (sequence derived from chicken N-CAM) and examined the aggregation behavior of isolated Ig domains in solution. The structure shows that Ig III adopts a canonical Ig fold, in which the beta strands ABED and A'GFCC' form two beta sheets that are linked by a disulfide bond. In contrast to the demonstrated aggregation of Ig III on solid supports, we were unable to demonstrate self-association of Ig III under any of a variety of solution conditions. The structure shows that the surface of Ig III is dominated by two large acidic patches, which may explain our failure to observe self-association in solution. To evaluate the involvement of the Ig I-Ig II interaction in cell-cell adhesion, we designed a point mutation in Ig I (F19S) that proved sufficient to abrogate the Ig I-Ig II interaction seen in solution. However, the introduction of this mutation into full-length N-CAM expressed in COS-7 cells failed to affect N-CAM-mediated cell-cell adhesion. The inability to observe Ig III self-association in solution, combined with the failure of the F19S mutation to affect N-CAM-mediated cell-cell adhesion, suggests that, although solution studies can give important insights into the structures of individual domains, the interactions observed in solution between the domains may not be representative of the interactions that occur on the cell surface.

Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity.

Although genetically engineered adenoviruses hold promise for the treatment of cancer, clinical trial reports have utilized intratumoral injection to date. To determine the feasibility of intravenous delivery of ONYX-015, an E1B-55kD gene-deleted replication selective adenovirus with demonstrated clinical safety and antitumoral activity following intratumoral injection, we performed a clinical trial in patients with metastatic solid tumors. ONYX-015 was infused intravenously at escalating doses of 2 x 10(10) to 2 x 10(13) particles via weekly infusion within 21-day cycles in 10 patients with advanced carcinoma metastatic to the lung. No dose-limiting toxicity was identified. Mild to moderate fever, rigors and a dose-dependent transient transaminitis were the most common adverse events. Neutralizing antibody titers significantly increased within 3 weeks in all patients. IL-6, gamma-IFN, TNF-alpha and IL-10 increased within 24 h following treatment. Evidence of viral replication was detectable in three of four patients receiving ONYX-015 at doses > or = 2 x 10(12) particles and intratumoral replication was confirmed in one patient. In conclusion, intravenous infusion of ONYX-015 was well tolerated at doses up to 2 x 10(13) particles and infection of metastatic pulmonary sites with subsequent intratumoral viral replication was seen. The intravenous administration of genetically altered adenovirus is a feasible approach.

Sodium nitroprusside compared with isoflurane-induced hypotension: the effects on brain oxygenation and arteriovenous shunting.

UNLABELLED: We compared sodium nitroprusside (SNP)-induced hypotension with 3% isoflurane-induced hypotension with regard to brain tissue oxygen pressure (PtO(2)), middle cerebral artery (MCA) blood flow, and cerebral arteriovenous shunting. Eight dogs were anesthetized with 1.5% isoflurane. After a craniotomy, a probe was inserted into the left frontoparietal brain cortex to mea-sure tissue gases and pH. Blood flow was measured in a secondary branch of the MCA by a flowprobe. Measurements were made during baseline 1.5% isoflurane, during 1.5% isoflurane and SNP-induced hypotension or 3% isoflurane-induced hypotension to a mean pressure of 60-65 mm Hg, and during continued treatment with SNP or 3% isoflurane with blood pressure support to baseline levels with phenylephrine. Shunting was calculated from arterial, sagittal sinus, and tissue (indicating capillary) oxygen content. During hypotension with SNP, PtO(2) decreased 50%, and shunting increased 50%. During hypotension with 3% isoflurane, PtO(2) and shunting did not change. Blood pressure support increased PtO(2) and MCA flow during both SNP and 3% isoflurane treatment. These results show that SNP is a cerebrovasodilator but that hypotension will decrease PtO(2), probably because of an increase in arteriovenous shunting and a decrease in capillary perfusion. IMPLICATIONS: We measured brain arteriovenous shunting and tissue oxygen pressure(PtO(2))during a 40% decrease in blood pressure induced by sodium nitroprusside (SNP)or 3% isoflurane. Large-dose isoflurane maintainedPtO(2) with no change in shunting. SNP infusion decreasedPtO(2) 50%and increased shunting 50%. This suggests that SNP-induced hypotension decreases PtO(2) because of a decrease in capillary perfusion.

Consciousness: the remembered present.

This chapter summarizes a theory of consciousness based on brain structure and dynamics. The theory centers around the notion of reentry--on-going recursive signaling across multiple reciprocally connected brain regions present mainly in the thalamocortical system. It recognized the fundamental beginnings provided by the complementary efforts of Ramón y Cajal and William James.

Internal initiation of translation of five dendritically localized neuronal mRNAs.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-kappa B activity in astrocytes.

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-kappaB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685-839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-kappaB to the nucleus. N-CAM also activated NF-kappaB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-kappaB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-kappaB and may be important in other N-CAM-mediated signaling.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides.

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells.

In higher eukaryotes, translation of some mRNAs occurs by internal initiation. It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs. In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5' leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs. When tested in the 5' untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells. Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells. The activity of the p150 leader was much greater than that of the YAP1 leader. The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5' leader sequence of the CLN3 mRNA in the intercistronic region. Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation. These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes. Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA. The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

Cerebral oxygen reactivity in the dog.

Brain tissue oxygen reactivity is a measure of the increase in tissue oxygen pressure (PtO2) relative to an increase in arterial oxygen pressure (PaO2). Clinical studies show that PtO2 reactivity is increased after cerebral injury. However, the impact of patient ventilation on these measures is not known. We determined whether changes in end tidal carbon dioxide pressure (ETCO2) would affect PtO2 reactivity in dogs. After a craniotomy, a Neurotrend probe that measures PtO2 was inserted into the cerebral cortex of eight dogs. PtO2 reactivity was measured at five concentrations of inspired oxygen (room air, 40%, 60%, 80%, 95%) at three levels of ETCO2 (20 mmHg, 40 mmHg, 60 mmHg) in random order. PtO2 reactivity at ETCO2 of 20 mmHg was 0.2 and increased to 0.3 when ETCO2 was 40 mmHg was 0.4 when ETCO2 was 60 mmHg (p < 0.05). These results show that PtO2 reactivity increases from hypocapnia to normocapnia. It is important to consider the ventilation state of each patient when evaluating PtO2.

Comparison of brain tissue and local cerebral venous gas tensions and pH.

Neurosurgical monitoring devices have recently become available which are capable of measuring cerebral tissue gas tensions and pH. Brain tissue sensors have not been conclusively demonstrated to correlate with other measurements of regional cerebral gas tensions or pH. The present study was undertaken to correlate sensor values for pO2, pCO2 and pH with blood samples taken concurrently from local cerebral veins. Adult mongrel dogs were anesthetized and a craniotomy was performed. A small gyral vein was isolated and cannulated. Adjacent to the venous catheter tip, a Neurotrend brain tissue probe was inserted in an intracortical location. Each subject received a sequence of manipulations in inspired oxygen and end tidal carbon dioxide conditions. Under each experimental condition, samples of arterial and gyral venous blood were obtained and blood gas analysis performed. Concurrent brain probe measurements of tissue pO2, pCO2 and pH were recorded. Statistical analysis determined that local tissue and cerebral venous blood values for pO2, pCO2 and pH were highly correlated (R(s) = 0.62-0.82; p < 0.001). This indicates that there exists a confirmable monotonic relationship between tissue values and conditions in the post-capillary venous bed. Tissue sensors such as the Neurotrend probe can offer reliable trend indications in brain tissue gas tensions and pH.

Isoflurane increases brain oxygen reactivity in dogs.

UNLABELLED: We tested the possibility that large-dose isoflurane will produce a loss of brain tissue oxygen regulation in dogs. A total of 12 dogs were anesthetized with isoflurane, a craniotomy was performed, and a probe was inserted to measure brain tissue oxygen pressure (PtO(2)), carbon dioxide, and pH. Baseline measures were made during 1.5% end-tidal isoflurane with 30% oxygen ventilation, followed by 95% oxygen ventilation. Six dogs (Group 1) were treated with 3% isoflurane and 30% oxygen, followed by a second oxygen challenge with 95% O(2). Six dogs (Group 2) received propofol to produce a similar suppression of the electroencephalogram as in Group 1, followed by 95% oxygen ventilation. Brain tissue oxygen reactivity was calculated by the increase in PtO(2) divided by the increase in arterial PO(2). During 1.5% isoflurane and propofol anesthesia, PtO(2) increased from 42 to 62 mm Hg with oxygen ventilation, and brain tissue oxygen reactivity was 0.14% per mm Hg(-1). Brain tissue oxygen reactivity did not change during propofol anesthesia. With 3% isoflurane, PtO(2) increased from 52 to 113 mm Hg and brain tissue oxygen reactivity was 0.36% per mm Hg(-1) (P: < 0.05). These results suggest that the cerebrovasodilator and vasoplegic effects of large-dose isoflurane attenuate brain oxygen regulation. IMPLICATIONS: We evaluated the ability of oxygen ventilation to increase brain tissue oxygen pressure in dogs anesthetized with 1.5% and 3% isoflurane and propofol. Increases in tissue oxygen were significantly greater during 3% isoflurane compared with 1.5% isoflurane and propofol.