Postmortem Subjects Research Articles

The mechanism of serum cell-free DNA release in postmortem subjects and novel markers to estimate the postmortem intervals.

Cell-free DNA (cfDNA) is DNA released from dying cells into the serum. The aim of the present study is to elucidate the mechanism of cell-free DNA (cfDNA) release in postmortem subjects by assaying Cytokeratin 18 (M30 and M65), cyclophilin A (CyPA), and myeloperoxidase (MPO) levels and to evaluate whether these levels are useful as markers for estimating postmortem intervals. Serum (n=54) was sampled from postmortem autopsied Japanese subjects. cfDNA was extracted and M30, M65, CyPA, and MPO in serum were assayed by enzyme-linked immunosorbent assay. Significantly higher serum levels of M30, M65, and MPO were observed in postmortem subjects than in living subjects. Although the difference was smaller, CyPA was also significantly higher in postmortem subjects than in living subjects. In addition, serum M30 and MPO levels were significantly correlated with cfDNA concentrations in postmortem subjects. Furthermore, M30 levels slightly increased according to the postmortem interval, and MPO levels at 2.5 days were significantly higher than those at <2 days. The result of regression analysis revealed a significant difference between M30/MPO levels and postmortem intervals. These findings suggested that elevated levels of cfDNA in postmortem subjects are released by apoptosis and neutrophils via NETosis and that M30 and MPO levels can be used as markers to estimate postmortem intervals.

Biophysical contrast sources for magnetic susceptibility and R2* mapping: A combined 7 Tesla, mass spectrometry and electron paramagnetic resonance study

Iron is the most abundant trace metal in the human brain and consistently shown elevated in prevalent neurological disorders. Because of its paramagnetism, brain iron can be assessed in vivo by quantitative MRI techniques such as R2* mapping and Quantitative Susceptibility Mapping (QSM). While Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has demonstrated good correlations of the total iron content to MRI parameters in gray matter, the relationship to ferritin levels as assessed by Electron Paramagnetic Resonance (EPR) has not been systematically analyzed. Therefore, we included 15 postmortem subjects (age: 26–91 years) which underwent quantitative in-situ MRI at 7 Tesla within a post-mortem interval of 24 h after death. ICP-MS and EPR were used to measure the total iron and ferritin content in 8 selected gray matter (GM) structures and the correlations to R2* and QSM were calculated. We found that R2* and QSM in the iron rich basal ganglia and the red nucleus were highly correlated with iron (R² > 0.7) and ferritin (R² > 0.6), whereas those correlations were lost in cortical regions and the hippocampus. The neuromelanin-rich substantia nigra showed a different behavior with a correlation with total iron only (R² > 0.5) but not with ferritin. Although qualitative results were similar for both qMRI techniques the observed correlation was always stronger for QSM than R2*. This study demonstrated the quantitative correlations between R2*, QSM, total iron and ferritin levels in an in-situ MRI setup and therefore aids to understand how molecular forms of iron are responsible for MRI contrast generation.

Post-mortem to ante-mortem facial image comparison for deceased migrant identification

The identification of deceased migrants is a global challenge that is exacerbated by migration distance, post-mortem conditions, access to ante-mortem data for comparison, inconsistent international procedures and lack of communication between arrival and origin countries. Due to low technology requirements, fast speed analysis and ease of transferring digital data, facial image comparison is particularly beneficial in those contexts, especially in challenging scenarios when this may be the only initial ante-mortem data available to identify the deceased. The Facial Identification Scientific Working Group (FISWG) professional guidelines for facial image comparison were developed for living facial appearance, and, therefore, a tailored protocol for the application of post-mortem to ante-mortem facial image comparison was proposed and evaluated in this research. The protocol was investigated via an inter-observer and an accuracy study, using 29 forensic cases (2001–2020) from the University of Milan, provided by the Laboratory of Forensic Anthropology and Odontology. In order to replicate a migrant identification scenario, each post-mortem subject was compared to all 29 ante-mortem targets (841 comparisons). The protocol guided the practitioner through stages of facial image comparison, from broad (phase 1) to more detailed (phase 3), eventually leading to a decision of ‘exclusion’ or ‘potential match’ for each post-mortem to ante-mortem case (phase 4). In phase 4, a support scale was also utilised to indicate the level of confidence in a potential match. Each post-mortem subject could be recorded with multiple potential matches. The protocol proved to be useful guide for facial image comparison, especially for less experienced practitioners and the inter-observer study suggested good reproducibility. The majority (82–96%) of ante-mortem subjects were excluded at the first stage of the protocol, and 71 full post-mortem to ante-mortem facial image comparisons were carried out. On average, two or three potential matches were recorded for each post-mortem subject. The overall accuracy rate was 85%, with the majority (79%) of ante-mortem non-targets correctly excluded from the identification process. An increased number and quality of available ante-mortem images produced more successful matches with higher levels of support. All potential matches involving non-targets received low levels of support, and for 73% of the post-mortem subjects, the ante-mortem target was the only recorded potential match. However, two ante-mortem targets were incorrectly excluded (one at the first stage of the protocol) and therefore changes to the protocol were implemented to mitigate these errors. A full protocol and a practical recording chart for practitioner use is included with this paper.

Lipidomic and Proteomic Insights from Extracellular Vesicles in Postmortem Dorsolateral Prefrontal Cortex Reveal Substance Use Disorder-Induced Brain Changes.

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to glial activation and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmann's area 9 (BA9) in postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n=4, SUD: n=4). The EVs were analyzed for physical properties (concentration, size, zeta potential, morphology) and subjected to integrative multi-omics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells as well as their effects on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between SUD and control groups. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD EVs. Proteomic analysis indicates downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB, and upregulation of C4A, C3, and ALB in SUD EVs. Gene ontology and protein-protein interactome analyses highlight functions such as cell motility, focal adhesion, and acute phase response signaling that is associated with the identified proteins. Both control and SUD EVs increased C3 and C4 mRNA expression in microglia, but only SUD EVs upregulated these genes in astrocytes. SUD EVs also significantly enhanced microglial migration in a wound healing assay.This study successfully isolated EVs from postmortem brains and used a multi-omics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD EVs and the distinct effects of EVs on glial cells suggest a crucial role in acute phase response signaling and neuroinflammation.

Serum detection of blood brain barrier injury in subjects with a history of stroke and transient ischemic attack

ObjectiveStroke and transient ischemic attack may have long-term negative effects on the blood-brain barrier (BBB) and promote endothelial inflammation, both of which could increase neurodegeneration and dementia risk beyond the cell death associated with the index event. MethodsSerum from 88 postmortem subjects in the Arizona Study of Aging and Neurodegenerative Disorders were analyzed by sandwich ELISA for specific biomarkers to investigate the effects of cerebrovascular accidents (CVAs) on BBB integrity and endothelial activation. Statistical analyses were performed using the Mann-Whitney U Test, Spearman rank correlation, and linear/logistic regressions adjusted for potential confounders; a P-value < .05 was considered significant for all analyses. ResultsSerum PDGFRẞ, a putative biomarker of BBB injury, was significantly increased in subjects with vs without a history of CVA who had similar cardiovascular risk factors (P < .01). This difference was stable after adjusting for age, hypertension, and other potential confounders in regression analysis (odds ratio, 27.02; 95% confidence interval, 2.61-411.7; P < .01). In addition, PDGFRẞ was positively associated with VCAM-1, a biomarker of endothelial inflammation (ρ = 0.42; P < .01). ConclusionsOur data suggest that patients with stroke or transient ischemic attack have lasting changes in the BBB. Still more, this demonstrates the utility of PDGFRẞ as a serum-based biomarker of BBB physiology, a potentially powerful tool in studying the role of the BBB in various neurodegenerative diseases and COVID infection sequelae. Clinical RelevanceOur data demonstrate the utility of serum PDGFRẞ, a putative biomarker of BBB integrity in the setting of stroke and TIA (CVA). A serum biomarker of BBB integrity could be a useful tool to detect early BBB damage and allow prospective work to study how such damage affects long-term neurodegenerative risk. Since BBB disruption occurs early in ADRD development, it could be monitored to help better understand disease progression and involvement of vascular pathways in ADRD.

Population Atlas Analysis of Emerging Brain Structural Connections in the Human Fetus.

A lack of in utero imaging data hampers our understanding of the connections in the human fetal brain. Generalizing observations from postmortem subjects and premature newborns is inaccurate due to technical and biological differences. To evaluate changes in fetal brain structural connectivity between 23 and 35 weeks postconceptional age using a spatiotemporal atlas of diffusion tensor imaging (DTI). Retrospective. Publicly available diffusion atlases, based on 60 healthy women (age 18-45 years) with normal prenatal care, from 23 and 35 weeks of gestation. 3.0 Tesla/DTI acquired with diffusion-weighted echo planar imaging (EPI). We performed whole-brain fiber tractography from DTI images. The cortical plate of each diffusion atlas was segmented and parcellated into 78 regions derived from the Edinburgh Neonatal Atlas (ENA33). Connectivity matrices were computed, representing normalized fiber connections between nodes. We examined the relationship between global efficiency (GE), local efficiency (LE), small-worldness (SW), nodal efficiency (NE), and betweenness centrality (BC) with gestational age (GA) and with laterality. Linear regression was used to analyze changes in GE, LE, NE, and BC throughout gestation, and to assess changes in laterality. The t-tests were used to assess SW. P-values were corrected using Holm-Bonferroni method. A corrected P-value <0.05 was considered statistically significant. Network analysis revealed a significant weekly increase in GE (5.83%/week, 95% CI 4.32-7.37), LE (5.43%/week, 95% CI 3.63-7.25), and presence of SW across GA. No significant hemisphere differences were found in GE (P = 0.971) or LE (P = 0.458). Increasing GA was significantly associated with increasing NE in 41 nodes, increasing BC in 3 nodes, and decreasing BC in 2 nodes. Extensive network development and refinement occur in the second and third trimesters, marked by a rapid increase in global integration and local segregation. 3 TECHNICAL EFFICACY: Stage 2.

Oral Cavity Fluid as an Alternative Postmortem Matrix: Comparison to Simultaneously Collected Blood and Urine Specimens.

In postmortem toxicology analysis, a variety of specimens consisting of fluids and tissues are often collected, each with an intrinsic value. Oral cavity fluid (OCF) is emerging as an alternative matrix in forensic toxicology for contributing to a diagnosis in postmortem cases; especially when blood is limited or not available. The aim of this study was to assess the analytical results obtained from OCF and compare them with blood, urine, and other traditional matrices collected from the same postmortem subjects. Of the 62 decedents studied (including 1 stillborn, 1 charred, and 3 decomposed subjects), 56 had quantifiable drugs and metabolites data in the OCF, blood, and urine. Notable findings were benzoylecgonine (24 cases), ethyl sulfate (23 cases), acetaminophen (21 cases), morphine (21 cases), naloxone (21 cases), gabapentin (20 cases), fentanyl (17 cases), and 6-acetylmorphine (15 cases), which were detected more frequently in OCF than in blood (heart, femoral, or body cavity) or urine. This study suggests that OCF is a suitable matrix for detecting and quantifying analytes in postmortem subjects compared with traditional matrices, particularly when other matrices are limited or difficult to collect because of body condition or putrefaction.

Evaluation of multi-channel phase reconstruction methods for quantitative susceptibility mapping on postmortem human brain

Quantitative Susceptibility Mapping (QSM) is an established Magnetic Resonance Imaging (MRI) technique with high potential in brain iron studies associated to several neurodegenerative diseases. Unlike other MRI techniques, QSM relies on phase images to estimate tissue's relative susceptibility, therefore requiring a reliable phase data. Phase images from a multi-channel acquisition should be reconstructed in a proper way. On this work it was compared the performance of combination of phase matching algorithms (MCPC3D-S and VRC) and phase combination methods based on a complex weighted sum of phases, considering the magnitude at different powers (k = 0 to 4) as the weighting factor. These reconstruction methods were applied in two datasets: a simulated brain dataset for a 4-coil array and data of 22 postmortem subjects acquired at a 7T scanner using a 32 channels coil. For the simulated dataset, differences between the ground truth and the Root Mean Squared Error (RMSE) were evaluated. For both simulated and postmortem data, the mean (MS) and standard deviation (SD) of susceptibility values of five deep gray matter regions were calculated. For the postmortem subjects, MS and SD were statistically compared across all subjects. A qualitative analysis indicated no differences between methods, except for the Adaptive approach on postmortem data, which showed intense artifacts. In the 20% noise level case, the simulated data showed increased noise in central regions. Quantitative analysis showed that both MS and SD were not statistically different when comparing k=1 and k=2 on postmortem brain images, however visual inspection showed some boundaries artifacts on k=2. Furthermore, the RMSE decreased (on regions near the coils) and increased (on central regions and on overall QSM) with increasing k. In conclusion, for reconstruction of phase images from multiple coils with no reference available, alternative methods are needed. In this study it was found that overall, the phase combination with k=1 is preferred over other powers of k.

A simple and reproducible measure of adipose depots with non-contrast post-mortem computed tomography

AbstractBackground and aimObesity is associated with an increase in different adipose depots. The anatomic distribution of internal adipose confers different risks. Recently, significant interest has emerged in the expansion of epicardial adipose tissue (EAT) as a mediator of adverse cardiovascular events. Often, post-mortem examination remains the best method of investigating morphological changes in health and disease. This study aimed to develop a simple, reproducible, and non-invasive protocol for the measurement of internal adiposity using post-mortem computed tomography (PMCT).Patients and methods101 consecutive post-mortem subjects underwent non-contrast computed tomography scans. Measurements were performed using the open-source software 3D Slicer by a non-expert researcher. An expert radiologist and cardiologist verified the abdominal and cardiac sites of adiposity, respectively. We aimed to develop a protocol to measure total EAT, sub-depots of EAT, extra-pericardial adipose, visceral and subcutaneous adipose, and suprasternal adipose.ResultsWe found excellent reproducibility for our measures of total EAT, anterior right atrial EAT, extra-pericardial adipose, and visceral adipose tissue, with intraclass correlations between 0.82 and 0.99 for each measure. Due to a lack of suitable anatomical boundaries, other sub-depots of EAT, including in the interventricular groove, were not reproducible.ConclusionsQuantification of total EAT and anterior right atrial EAT are readily reproducible using 3D Slicer on post-mortem CT. They can be reliably measured by non-expert researchers with a small amount of training, and therefore be used to investigate morphological changes in adiposity in health and disease.

Differential expression of AMPK subunit isoforms in subcutaneous adipose tissue of post-mortem subjects with BMI&gt;25kg/m2

AMP-activated protein kinase (AMPK) is a heterotrimeric complex that is made up of α-, β- and γ- subunits isoforms making up various isomers. AMPK has been shown to improve catabolic ATP-generating pathways, making it a promising therapeutic target. Studies have shown that different AMPK isomers are predominant in different tissues. However, determination of the predominant isomer in adipose tissue remains obscure. The goal of this study is to identify the AMPK isomers present in human subcutaneous adipose tissues comparing between lean and overweight/obese subjects. Total RNA from post-mortem subcutaneous adipose tissue was extracted and its quantity and integrity were evaluated using a bioanalyzer before being reverse transcribed to cDNA. Then, by using SYBR-green as a fluorophore detection, quantitative real-time PCR was performed. The relative expression of each target gene was calculated using the comparative 2-∆∆Ct method, and the expression levels of target genes were normalized to that of GAPDH, RPLP0, and HPRT1. Mean differences of AMPK subunits mRNA level for both lean and overweight/obese subjects were determined by Mann-Whitney for two-sample comparisons using SPSS 26.0 (SPSS Inc., USA). This study revealed that in overweight/obese subjects, the gene expression of α1-AMPK was upregulated (2.5-fold) whilst α2-AMPK was downregulated (0.5-fold) when compared to lean controls (p=0.044). There were no differences observed between the β and γ subunits in both lean and overweight/obese subjects. Findings from this study suggests that α1- and α2-AMPK gene expression plays an important role in overweight/obese subjects. Future studies to explore the AMPK gene expressions in visceral adipose tissue and among underweight subjects would further close this knowledge gap.

Nox2 underpins microvascular inflammation and vascular contributions to cognitive decline

Chronic microvascular inflammation and oxidative stress are inter-related mechanisms underpinning white matter disease and vascular cognitive impairment (VCI). A proposed mediator is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2), a major source of reactive oxygen species (ROS) in the brain. To assess the role of Nox2 in VCI, we studied a tractable model with white matter pathology and cognitive impairment induced by bilateral carotid artery stenosis (BCAS). Mice with genetic deletion of Nox2 (Nox2 KO) were compared to wild-type (WT) following BCAS. Sustained BCAS over 12 weeks in WT mice induced Nox2 expression, indices of microvascular inflammation and oxidative damage, along with white matter pathology culminating in a marked cognitive impairment, which were all protected by Nox2 genetic deletion. Neurovascular coupling was impaired in WT mice post-BCAS and restored in Nox2 KO mice. Increased vascular expression of chemoattractant mediators, cell-adhesion molecules and endothelial activation factors in WT mice post-BCAS were ameliorated by Nox2 deficiency. The clinical relevance was confirmed by increased vascular Nox2 and indices of microvascular inflammation in human post-mortem subjects with cerebral vascular disease. Our results support Nox2 activity as a critical determinant of VCI, whose targeting may be of therapeutic benefit in cerebral vascular disease.

Molecular characterization of depression trait and state.

Major depressive disorder (MDD) is a brain disorder often characterized by recurrent episode and remission phases. The molecular correlates of MDD have been investigated in case-control comparisons, but the biological alterations associated with illness trait (regardless of clinical phase) or current state (symptomatic and remitted phases) remain largely unknown, limiting targeted drug discovery. To characterize MDD trait- and state-dependent changes, in single or recurrent depressive episode or remission, we generated transcriptomic profiles of subgenual anterior cingulate cortex of postmortem subjects in first MDD episode (n = 20), in remission after a single episode (n = 15), in recurrent episode (n = 20), in remission after recurring episodes (n = 15) and control subject (n = 20). We analyzed the data at the gene, biological pathway, and cell-specific molecular levels, investigated putative causal events and therapeutic leads. MDD-trait was associated with genes involved in inflammation, immune activation, and reduced bioenergetics (q < 0.05) whereas MDD-states were associated with altered neuronal structure and reduced neurotransmission (q < 0.05). Cell-level deconvolution of transcriptomic data showed significant change in density of GABAergic interneurons positive for corticotropin-releasing hormone, somatostatin, or vasoactive-intestinal peptide (p < 3 × 10-3). A probabilistic Bayesian-network approach showed causal roles of immune-system-activation (q < 8.67 × 10-3), cytokine-response (q < 4.79 × 10-27) and oxidative-stress (q < 2.05 × 10-3) across MDD-phases. Gene-sets associated with these putative causal changes show inverse associations with the transcriptomic effects of dopaminergic and monoaminergic ligands. The study provides first insights into distinct cellular and molecular pathologies associated with trait- and state-MDD, on plasticity mechanisms linking the two pathologies, and on a method of drug discovery focused on putative disease-causing pathways.

Comparison of serum cell-free DNA between postmortem and living samples

Cell-free DNA (cfDNA) originates from apoptotic and/or necrotic cells. Few reports are available that examine cfDNA from postmortem samples. Therefore, this study investigated differences between postmortem and biogenic subjects in concentration and fragment distribution of serum cfDNA. We also clarified features of serum cfDNA in postmortem subjects. The results revealed that postmortem subjects had significantly higher cfDNA concentrations than healthy controls and patients with cardiac disease. Serum cfDNA concentrations increased slightly with postmortem interval in subjects who died of asphyxia, and they were slightly higher in subjects who died from internal vs. external causes. Microchip electrophoresis of serum cfDNA revealed a fragment larger than 10,000 bp in only two postmortem subjects; we speculate that the fragment may have originated from necrotic cells. A relatively high concentration of one 150–200 bp fragment was characteristic of postmortem samples. This fragment may have been derived from apoptosis or other processes. We also observed ladder fragments in some subjects who died from external causes. Although additional research is needed for verification, serum cfDNA concentrations and fragment patterns possibly be used as a tool to estimate postmortem intervals and cause of death.

45698 Molecular Signatures of Cocaine Neurotoxicity in Human Brain Models

ABSTRACT IMPACT: This project will use human neuron models and bioinformatics techniques to elucidate mechanisms of cocaine neurotoxicity, which will allow treatments to be developed for minimizing or preventing neurological damage caused by cocaine abuse and overdose. OBJECTIVES/GOALS: The goals of this project are to identify genes and gene networks altered by cocaine exposure in neurons (short term), and to use these pathways to understand mechanisms of cocaine neurotoxicity for the establishment of therapeutic targets (long term). METHODS/STUDY POPULATION: To study the molecular effects of cocaine, we generated preliminary proteomics and next-generation RNA sequencing (RNAseq) data from human postmortem prefrontal cortex (Broadmann area 9 or BA9) of 12 cocaine overdose subjects and 17 controls. Future directions for this project include RNAseq analysis of neuronal nuclei sorted from human postmortem BA9 and a human induced pluripotent stem cell-derived neuron (hiPSN) model of cocaine exposure from the same postmortem subjects from whom we have brain samples. RESULTS/ANTICIPATED RESULTS: We found alterations in neuronal synaptic protein levels and gene expression, including the serotonin transporter SLC6A4, and synaptic proteins SNAP25, SYN2, SYNGR3. Pathway analysis of our results revealed alterations in specific pathways involved with neuronal function including voltage-gated calcium channels, and GABA receptor signaling. In the future, we expect to see an enhancement in neuron-specific gene expression signatures in our sorted neuronal nuclei and our hiPSN model of cocaine exposure. The hiPSN model will help elucidate which effects are due to acute versus chronic exposure of cocaine. DISCUSSION/SIGNIFICANCE OF FINDINGS: Transcriptomic signatures found with this analysis can help us understand mechanisms of cocaine neurotoxicity in human neurons. With this work and future proposed studies, we can discover targetable molecular pathways to develop drugs that can reduce or reverse cocaine-related impairment.

No Detectable Electroencephalographic Activity After Clinical Declaration of Death Among Tibetan Buddhist Meditators in Apparent Tukdam, a Putative Postmortem Meditation State.

Recent EEG studies on the early postmortem interval that suggest the persistence of electrophysiological coherence and connectivity in the brain of animals and humans reinforce the need for further investigation of the relationship between the brain’s activity and the dying process. Neuroscience is now in a position to empirically evaluate the extended process of dying and, more specifically, to investigate the possibility of brain activity following the cessation of cardiac and respiratory function. Under the direction of the Center for Healthy Minds at the University of Wisconsin-Madison, research was conducted in India on a postmortem meditative state cultivated by some Tibetan Buddhist practitioners in which decomposition is putatively delayed. For all healthy baseline (HB) and postmortem (PM) subjects presented here, we collected resting state electroencephalographic data, mismatch negativity (MMN), and auditory brainstem response (ABR). In this study, we present HB data to demonstrate the feasibility of a sparse electrode EEG configuration to capture well-defined ERP waveforms from living subjects under very challenging field conditions. While living subjects displayed well-defined MMN and ABR responses, no recognizable EEG waveforms were discernable in any of the tukdam cases.

4141 Molecular Signatures of Cocaine Toxicity in Postmortem Human Brain and Neurons

OBJECTIVES/GOALS: The goal of this project is to identify new therapeutic targets and biomarkers to treat or prevent cocaine toxicity by investigating proteomic, transcriptomic and epigenetic signatures of cocaine exposure in human subjects. METHODS/STUDY POPULATION: Cocaine is a highly addictive neurotoxic substance, and it is estimated that 1.9 million Americans are current users of cocaine. To study the molecular effects of cocaine, we generated preliminary proteomics and next-generation RNA sequencing (RNAseq) data from human postmortem dorsolateral prefrontal cortex (Broadmann area 9 or BA9) of 12 cocaine-exposed subjects and 17 controls. Future directions for this project include RNAseq and DNA methylation analysis of neuronal nuclei sorted from human postmortem BA9 and a human induced pluripotent stem cell-derived neuron (hiPSN) model of cocaine exposure from the same postmortem subjects from whom we have brain samples. RESULTS/ANTICIPATED RESULTS: We found alterations in neuronal synaptic protein levels and gene expression, including the serotonin transporter SLC6A4, and synaptic proteins SNAP25, SYN2, SYNGR3. Pathway analysis of our results revealed alterations in specific pathways involved with neuronal function including voltage-gated calcium channels, and GABA receptor signaling. In the future, we expect to see an enhancement in neuron-specific gene expression signatures in our sorted neuronal nuclei and our hiPSN model of cocaine exposure. The hiPSN model will help elucidate which effects are due to acute versus chronic exposure of cocaine. DISCUSSION/SIGNIFICANCE OF IMPACT: Neuronal signatures found with this analysis can help us understand mechanisms of cognitive decline in long-term cocaine users as well as the acute effects on the brain of cocaine taken in overdose. With this work and future proposed studies, we can discover novel clinical biomarkers for cocaine neurotoxicity in patients with cocaine use disorder and determine readouts for future therapeutic development on cocaine addiction and overdose.

Morphological differences between coastal bottlenose dolphin (Tursiops aduncus) populations identified using non-invasive stereo-laser photogrammetry

Obtaining morphometric data on free-ranging marine megafauna is difficult, as traditional methods rely on post-mortem or live-capture techniques. We linked stereo-laser photogrammetry with long-term demographic data to compare length-at-age (LaA) growth curves of two well-studied populations of Indo-Pacific bottlenose dolphins (Tursiops aduncus) in south-western (SW) and Shark Bay (SB), mid-western Australia. First, we determined the relationship between total length (TL) and blowhole-to-dorsal fin (BH-DF) length from post-mortem subjects (R2 = 0.99, n = 12). We then predicted TL from laser-derived BH-DF measurements of 129 and 74 known-age individuals in SW and SB, respectively. Richards growth models best described our LaA data. While birth length (103–110 cm) was similar between study regions, TL estimates at 1, 3, 12, and 25 years differed significantly (p < 0.001). Asymptotic length of adult males (SW = 246 cm, SB = 201 cm) and females (SW = 244 cm, SB = 200 cm) also differed significantly. Morphotypic variations likely reflect regional adaptations to local water temperatures, with the temperate SW having cooler waters than sub-tropical SB. We demonstrate the effectiveness of a non-invasive technique to understand ecological, demographic and life-history characteristics of long-lived marine megafauna, which are critical parameters for informing conservation and management actions.

Neurofibrillary Pathology in the Infundibular Nucleus in Relation to Age and Abnormal Hormone Levels

Abstract&#x0D; Objective: to determine whether the decline of testosterone during ageing would make this nucleus more vulnerable for NF changes (i.e. hyperphosphorylated-tau) in men, or that the decline of estrogens in the postmenopausal period would protect the infundibular nucleus in women.&#x0D; Methods: We investigated the infundibular nucleus in postmortem subjects. Brain materials obtained from 29 subjects in the Netherlands Brain Bank were further classified as control subjects and subjects with abnormal hormone conditions. Procedures consisted of tissue collection, immunochemical staining, and analysis of the staining intensity. Results then were collected and concluded using observational methods.&#x0D; Results: Elderly male subjects with low testosterone conditions showed more severe NF changes in the infundibular nucleus than postmenopausal women. The occurrence of NF changes in elderly subjects was generally accompanied by the presence of basket-like nerve terminals staining for ERβ.&#x0D; Conclusion: The sex difference in NF changes in the infundibular nucleus in the elderly is due to hyperphosphorylated-tau induction in low testosterone and ageing condition in men, while in postmenopausal women the declining estrogen levels seem to protect against NF changes in this brain area.&#x0D; Keywords: ageing, estrogen, hyperphosphorylated-tau, infundibular nucleus, testosterone&#x0D; &#x0D; Abstrak&#x0D; Tujuan: untuk menentukan apakah penurunan level testosteron selama proses penuaan menyebabkan nukleus infundibularis menjadi lebih rentan terhadap perubahan neurofibrilar (NF) (misalnya hyperphosphorylated-tau) pada laki-laki atau apakah penurunan level estrogen selama masa pasca-menopause memiliki efek protektif terhadap nukleus infundibular pada perempuan.&#x0D; Metode: Peneliti memeriksa nukleus infundibular pada subjekpost-mortem. Materi berupa jaringan otak dari 29 subjek dari Netherlands Brain Bank lebih lanjut diklasifikasikan sebagai subjek kontrol dan subjek dengan kondisi hormon abnormal. Prosedur terdiri dari pengumpulan jaringan, pewarnaan dengan teknik imunohistokimia, dan analisis dari intensitas pewarnaan. Hasil yang didapat kemudian dikumpulkan dan disimpulkan sesuai dengan metode observasional.&#x0D; Hasil: Subjek laki-laki lanjut usia dengan testosteron rendah menunjukkan perubahan NF yang lebih buruk pada nukleus infundibular dibandingkan dengan wanita postmenopause. Kejadian perubahan NF pada subjek lanjut usia secara umum diikuti oleh munculnya pewarnaan pada ujung saraf berbentuk basket-like yang positif untuk Erβ.&#x0D; Kesimpulan:Perbedaan jenis kelamin terkait perubahan NF pada nukleus infundibular pada subjek lanjut usia terjadi akibat induksi hiperfosforilasi taupada kondisi testosteron yang rendah yang dikombinasi oleh proses penuaan pada pria. Sedangkan pada perempuan pascamenopause, penurunan level estrogen menunjukkan efek protektif terhadap perubahan NF pada area otak ini.&#x0D; Kata kunci: estrogen, hiperfosforilasi protein tau, nukleus infundibularis, penuaan, testosteron

Expression of immune markers in post-mortem brain subjects of major depression and suicidality

Expression of immune markers in post-mortem brain subjects of major depression and suicidality

Oral Cavity Fluid as an Investigative Approach for Qualitative and Quantitative Evaluations of Drugs in Postmortem Subjects.

A relatively overlooked aspect of forensic science is the potential of oral cavity fluid for contributing to a forensic diagnosis. Although traditional specimens, like blood and urine, are routinely evaluated for forensic toxicology testing, fluid from the oral cavity has not been investigated as a matrix in postmortem cases. Our laboratory developed and validated qualitative and quantitative analytical methods for determining 47 medicinal and illicit drugs from oral cavity fluid. These developed methods aimed to compare results from liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of oral cavity fluid to those of traditional matrices collected from the same postmortem subjects. Of 34 cadavers studied, 32 (including two decomposed and two drowned subjects) had detectable and quantifiable drugs in the oral cavity fluid and/or blood, urine, bile, vitreous fluid and/or liver tissue. The most significant finding was that 6-acetylmorphine (6-AM) was detected more frequently in oral cavity fluid (11 cases) than in blood and urine combined (6 cases). Compounds with a short window of detection, like the heroin metabolite, 6-AM and even heroin, could be detected more readily in oral cavity fluid than in urine. In 2017, the incidence of heroin-related overdose deaths increased to 15,958. Those data have shed light on the practicality of testing oral cavity fluid postmortem and its significance in forensic toxicology. In conclusion, this study showed that oral cavity fluid could be useful for detecting and quantifying drugs in postmortem subjects; moreover, oral cavity fluid may be particularly suitable when other matrices are limited or difficult to collect, due to body condition or putrefaction.