Postmortem Material Research Articles

Electrochemical aptasensor for 2-amino-2-thiazoline-4-carboxylic acid (ATCA), a metabolite for cyanide poisoning

An alternative biomarker for assessing the cyanide levels in postmortem materials is crucial for investigating acute cyanide intoxication. Herein, an aptamer–ligand biorecognition system with high specificity was developed to detect acute cyanide poisoning via its secondary metabolite, 2-amino-2-thiazoline-4-carboxylic acid (ATCA). Potential aptamers were screened from a random library of 66-base single-stranded DNA using GO-SELEX, with individual aptamers being identified through single-stranded DNA sequencing. Molecular docking was employed to predict the affinity of these aptamers toward ATCA and selected counter-targets; these predictions were confirmed using thermodynamic analysis with an isothermal titration calorimeter. Owing to its label-free biomolecular binding interactions, Apt46 exhibited the highest affinity against ATCA and notable selectivity against structurally similar counter-targets. Thus, an amino-tagged Apt46 binding aptamer was attached to a carbon electrode modified with EDC–NHS-activated graphene oxide. The binding of Apt46 to ATCA was quantified by measuring current changes using differential pulse voltammetry. The aptasensor achieved a detection limit of 0.05 µg/mL and demonstrated suitability for detecting ATCA across various biological matrices, with the high recovery percentages ranging from 92.29 to 114.22%. Overall, the proposed ATCA aptasensor is promising for identifying ATCA metabolites in cases of acute cyanide exposure.

(Invited) Catalyst and Anode Design for Durable Alkaline-Membrane Electrolysis

Commercialized membrane electrolyzers use acidic proton exchange membranes (PEMs). These systems offer high performance but require the use of expensive precious-metal catalysts such as IrO2 and Pt that are nominally stable under locally acidic conditions. Alkaline-exchange-membrane (AEM) electrolyzers in principle offer the performance of PEM electrolyzers with the ability to use earth-abundant catalysts and inexpensive bipolar plate materials. I will highlight our fundamental work in understanding the chemical and electrochemical processes in earth-abundant water-oxidation catalysts over the past decade and we are using that understanding to drive progress in high-performance AEM electrolyzers.Baseline systems operate at 1 A·cm-2 in pure water feed at < 1.9 V at a moderate temperature of ~70 °C using either IrO2 or Co3O4 anode catalyst layers, PiperION alkaline ionomers, and stainless-steel porous transport layers. These devices, however, degrade rapidly compared to PEM electrolyzers which we link to chemical and structural changes in the ionomer-catalyst reactive zone using a combination of integrated reference-electrode device architectures, impedance, and cross-sectional and post-mortem materials analysis. We further discover that dynamic Fe-based OER catalysts – that have world-record performance in traditional liquid alkaline electrolyzer systems – perform poorly with enhanced degradation rates in alkaline membrane electrolysis, illustrating fundamentally different chemical design principles for OER catalysts.Given this baseline understanding, I will end with new chemical strategies we have developed to mitigate degradation and enhance performance using novel ionomers, passivated electrolyte-catalyst interfacial architectures, and specifically designed multicomponent anode oxygen catalysts to target stable performance without supporting soluble electrolyte: <10 μV/h degradation at currents > 2 A cm-2 and cell voltages < 2V.

High-speed perforation of IN718 plates by spherical TC4 Ti alloy projectiles: Experiments and modeling

Data and knowledge on ballistic performance of Inconel-718 (IN718) alloy is critical for evaluating the resistance of turbine blades against foreign object impacts in aeronautical and astronautical industries. Ballistic perforation experiments (impact velocity 480 ms−1 to 1340 ms−1) with TC4 spherical projectiles (5-mm-diameter) are conducted on IN718 target plates (2-mm-thick) in this work, along with high-speed photography. The IN718 plates show multiple deformation and failure modes (bulging, dishing, petaling and plugging). The ballistic limit velocity for the investigated projectile/target combination is 823 ms−1. Postmortem material characterizations (optical photography, scanned electron microscopy and energy dispersive spectrometry) are conducted on recovered projectiles and bullet hole inner walls, and the observations indicate adiabatic shearing, surface melting and abrasion of projectile material. A finite element model based on the Johnson–Cook constitutive model and failure criterion is established, and reproduces the ballistic experiments. Dimensionless analyses are conducted on the dimensions of postmortem projectiles and bullet holes, and the projectile residual velocity/mass. On the basis of experimental observations and theoretical analysis of the projectile dynamics, the whole perforation process is divided into three stages (the entering, cratering and plugging stages). A multi-stage analytical model on perforation is then developed considering the effects of cavity expansion, projectile deformation and abrasion, and can well predict the projectile dynamics during entering and cratering stages.

Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

High-temperature tensile strength of alumina-spinel refractory by Brazilian test at 1200°C coupled with DIC in-situ monitoring

This study investigated for the first time the corrosion effect of Al2O3-MgAl2O4 refractory by steel ladle slag using a DIC-coupled Brazilian test at 1200 °C. Three types of alumina-spinel materials were used in the test, namely, original non-infiltrated refractory, slag-infiltrated at 1450 °C as well as postmortem refractory after 69 cycles of work in the sidewalls of steel ladle. Lab infiltration was conducted at 1450 °C using CaO-Al2O3-SiO2-based ladle slag and its analog enriched with manganese (10% MnO). The tensile strength of postmortem material at 1200 °C was the highest at 10.7 MPa, and it was 52% and 70% higher than for reference and MnO-enriched slag, respectively, and 80% higher than for original refractory. The significantly raised tensile strength of slag-infiltrated materials vs. original non-infiltrated brick was directly related to the densification of microstructure by corrosion products (mainly CA6, CA2, Fe) which caused the infiltrated materials to be stiffer and with a more brittle nature to fracture.

Analysis of cases of suicide by self-immolation in the post-mortem material of the Department of Forensic Medicine in Cracow.

Analysis of self-immolation cases and distribution of the resulting burns and their degree. The study included 16 cases from the Department of Forensic Medicine, Jagiellonian University Medical College in Cracow from 2000-2022 in which the cause of death was self-immolation. Based on the analysis of photographs and autopsy reports, drawings were made showing the exact distribution and nature of the injuries, moreover, the approximate percentage of body surface area affected was determined as well as the frequency of involvement of specific areas of the body, and the presence of previous diseases and mental disorders including previous suicide attempts. 81% of victims were male. Two age groups were predominant among the cases analyzed, namely, individuals around the age of 20, and those between 50 and 60 years of age. 44% of the deceased had burns exceeding 80% of total body surface. The most frequently involved body areas were the extremities and chest as well as head and neck. Fourth-degree burns were most prevalent on the head and neck, third-degree burns prevailed on the upper and lower extremities, second-degree burns were mostly found on the chest, and first-degree burns - on the lower extremities. There were no cases of fourth-degree burns of the buttocks. 38% of the subjects had a history of substance abuse, 56% suffered from mental illnesses, whereas 31% attempted suicide in the past. The distribution of burns in self-immolation cases is inhomogeneous. The most frequently affected area was the head, neck, chest and extremities, most likely due to victims dousing themselves with a flammable substance from the top of the head through the chest. In all cases, the immediate cause of death was burn disease, regardless of the size of the body surface area affected by the burns. The majority of victims had a history of mental illness, substance abuse or suicide attempts.

Head-to-head comparison of [18F]-Flortaucipir, [18F]-MK-6240 and [18F]-PI-2620 postmortem binding across the spectrum of neurodegenerative diseases

We and others have shown that [18F]-Flortaucipir, the most validated tau PET tracer thus far, binds with strong affinity to tau aggregates in Alzheimer's (AD) but has relatively low affinity for tau aggregates in non-AD tauopathies and exhibits off-target binding to neuromelanin- and melanin-containing cells, and to hemorrhages. Several second-generation tau tracers have been subsequently developed. [18F]-MK-6240 and [18F]-PI-2620 are the two that have garnered most attention. Our recent data indicated that the binding pattern of [18F]-MK-6240 closely parallels that of [18F]-Flortaucipir. The present study aimed at the direct comparison of the autoradiographic binding properties and off-target profile of [18F]-Flortaucipir, [18F]-MK-6240 and [18F]-PI-2620 in human tissue specimens, and their potential binding to monoamine oxidases (MAO). Phosphor-screen and high resolution autoradiographic patterns of the three tracers were studied in the same postmortem tissue material from AD and non-AD tauopathies, cerebral amyloid angiopathy, synucleopathies, transactive response DNA-binding protein 43 (TDP-43)-frontotemporal lobe degeneration and controls. Our results show that the three tracers show nearly identical autoradiographic binding profiles. They all strongly bind to neurofibrillary tangles in AD but do not seem to bind to a significant extent to tau aggregates in non-AD tauopathies pointing to their limited utility for the in vivo detection of non-AD tau lesions. None of them binds to lesions containing β-amyloid, α-synuclein or TDP-43 but they all show strong off-target binding to neuromelanin and melanin-containing cells, as well as weaker binding to areas of hemorrhage. The autoradiographic binding signals of the three tracers are only weakly displaced by competing concentrations of selective MAO-B inhibitor deprenyl but not by MAO-A inhibitor clorgyline suggesting that MAO enzymes do not appear to be a significant binding target of any of them. These findings provide relevant insights for the correct interpretation of the in vivo behavior of these three tau PET tracers.

Integrative proteomics highlight presynaptic alterations and c-Jun misactivation as convergent pathomechanisms in ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease mainly affecting upper and lower motoneurons. Several functionally heterogeneous genes have been associated with the familial form of this disorder (fALS), depicting an extremely complex pathogenic landscape. This heterogeneity has limited the identification of an effective therapy, and this bleak prognosis will only improve with a greater understanding of convergent disease mechanisms. Recent evidence from human post-mortem material and diverse model systems has highlighted the synapse as a crucial structure actively involved in disease progression, suggesting that synaptic aberrations might represent a shared pathological feature across the ALS spectrum. To test this hypothesis, we performed the first comprehensive analysis of the synaptic proteome from post-mortem spinal cord and human iPSC-derived motoneurons carrying mutations in the major ALS genes. This integrated approach highlighted perturbations in the molecular machinery controlling vesicle release as a shared pathomechanism in ALS. Mechanistically, phosphoproteomic analysis linked the presynaptic vesicular phenotype to an accumulation of cytotoxic protein aggregates and to the pro-apoptotic activation of the transcription factor c-Jun, providing detailed insights into the shared pathobiochemistry in ALS. Notably, sub-chronic treatment of our iPSC-derived motoneurons with the fatty acid docosahexaenoic acid exerted a neuroprotective effect by efficiently rescuing the alterations revealed by our multidisciplinary approach. Together, this study provides strong evidence for the central and convergent role played by the synaptic microenvironment within the ALS spinal cord and highlights a potential therapeutic target that counteracts degeneration in a heterogeneous cohort of human motoneuron cultures.

Effect of Si layer thickness on the cycling stability and aging behavior of Li-ion capacitors with micrometer-sized Si anodes

Si is utilized as the active anode material of lithium-ion capacitors (LICs) to increase their energy and power densities. The main drawback of industrial Si-based LICs is their insufficient cycling stability resulting from the limited life of Si anodes. In this study, cell design parameters required for 10,000 stable cycles were determined using inexpensive unmodified 2 μm Si particles and a polyimide binder. Three cathode-to-anode capacity ratios were investigated by varying the thickness of a Si coating layer at a constant mass of the cathode fabricated from activated carbon (AC). The thickest Si coating with a thickness of 48 μm resulted in the anode/cathode capacity ratio of 56.4, which was calculated based on the apparent Si specific capacity of 3000 mAh g−1 and AC specific capacity of 60 mAh g−1. Conversely, the thinnest Si coating with a thickness of 10 μm exhibited a capacity ratio of 14.2 and highest energy density of 97.4 Wh kg−1 at a power density of ∼1 kW kg−1 during the first cycle, and the highest cycling stability corresponding to a retention of 78.3% after 30,000 cycles. Additionally, the aging behavior of the produced anodes and cathodes was examined via postmortem material characterization.

Assessment of misdiagnosis in small animal intensive care patients using the Modified Goldman criteria

The postmortem examination can be used as a means of quality control for clinical diagnoses. A retrospective study on 300 dogs and cats that had been admitted to a small animal intensive care unit was performed comparing the clinical and postmortem findings, using the Modified Goldman criteria. All patient files were reevaluated for clinical diagnoses and all postmortem material was reevaluated for pathological diagnoses. After this, the Modified Goldman criteria were applied to score the discrepancies between them, and factors associated with the occurrence of an undiagnosed major unexpected finding were analyzed. The postmortem examination revealed additional findings in 65% of the cases. Major discrepancies, defined as those affecting treatment and possibly outcome of the patient, were present in 21.3% of the cases. The most frequently missed diagnoses detected at necropsy were pneumonia of various etiologies, meningitis/meningoencephalitis, myocarditis and generalized vasculitis. A shorter ICU stay was associated with increased odds of a major discrepancy. Conditions affecting the urinary or gastrointestinal system were negatively associated with major discrepancy.

Suicide of minors—Retrospective 22-year analysis of the post-mortem and autopsy material of the Institute of Forensic Medicine Rostock

ZusammenfassungHintergrundInsgesamt treten Suizide bei Kindern und Jugendlichen zwar selten auf, sie stellen jedoch die zweithäufigste Todesursache nach Unfällen bei unter 18-Jährigen dar. Während die Unfalltodeszahlen von Kindern und Jugendlichen seit Jahren sinken, bleibt die Suizidrate nahezu konstant.MethodikIm Rahmen dieser Untersuchung wurden Fälle von Suiziden bei Kindern und Jugendlichen, die jünger als 18 Jahre waren, im Zeitraum von Januar 2001 bis Dezember 2022 retrospektiv ausgewertet.ErgebnisseWährend des betrachteten Zeitraumes konnten 13 Suizide von Kindern und Jugendlichen in die Erhebung eingeschlossen werden, wobei in 10 Fällen eine gerichtlich angeordnete Obduktion durchgeführt wurde. Das Alter der 8 betroffenen Mädchen und 5 betroffenen Jungen erstreckte sich vom 12. bis zum 17. Lebensjahr. Die Kinder und Jugendlichen verstarben am häufigsten durch Erhängen, das in einem Fall kurzzeitig überlebt wurde, sowie an den Folgen eines Polytraumas. Andere Todesursachen waren Intoxikationen mittels unterschiedlicher Substanzen sowie ein Kopfschuss. In 3 Fällen war eine Depression vorbekannt.DiskussionZwar sind Suizide im Kindes- und Jugendalter selten, dennoch stellen sie für beteiligte Personen immer ein tragisches Ereignis dar. Vor allem die Unterscheidung zwischen einem Suizid und einem Unfalltod kann schwierig sein. Hierbei kann insbesondere eine rechtsmedizinische Leichenschau am Ereignisort mit anschließender Obduktion entscheidend zur Aufklärung des Falls beitragen.

Astrocytes: Dissecting Their Diverse Roles in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders often co-occurring in the same patient, a feature that suggests a common origin of the two diseases. Consistently, pathological inclusions of the same proteins as well as mutations in the same genes can be identified in both ALS/FTD. Although many studies have described several disrupted pathways within neurons, glial cells are also regarded as crucial pathogenetic contributors in ALS/FTD. Here, we focus our attention on astrocytes, a heterogenous population of glial cells that perform several functions for optimal central nervous system homeostasis. Firstly, we discuss how post-mortem material from ALS/FTD patients supports astrocyte dysfunction around three pillars: neuroinflammation, abnormal protein aggregation, and atrophy/degeneration. Furthermore, we summarize current attempts at monitoring astrocyte functions in living patients using either novel imaging strategies or soluble biomarkers. We then address how astrocyte pathology is recapitulated in animal and cellular models of ALS/FTD and how we used these models both to understand the molecular mechanisms driving glial dysfunction and as platforms for pre-clinical testing of therapeutics. Finally, we present the current clinical trials for ALS/FTD, restricting our discussion to treatments that modulate astrocyte functions, directly or indirectly.

A fatal case of poisoning with a cathinone derivative: α-PiHP and its post-mortem distribution in body fluids and organ tissues.

New psychoactive substances (NPS) continue to appear on the drug market and alpha-pyrrolidinoisohexanophenone (α-PiHP) is one of the most popular cathinone derivatives. In this paper, we report a case of death caused by α-PiHP. Based on the toxicological results of the studied case along with autopsy, histopathological findings and crime-scene information, fatal intoxication with α-PiHP was accepted as the final cause of death. α-PiHP and its metabolite (OH-α-PiHP) were detected and quantified in all post-mortem materials (blood collected from the heart, the femoral vein and the dural venous sinuses, vitreous humour, cerebrospinal fluid, cerebral cortex, brainstem, cerebellum, bile, liver, kidney, heart, pancreas, spleen, thyroid gland, lung, adipose tissue, stomach and intestine). To date, this is the first case of determination of α-PiHP and its metabolite in post-mortem specimens. In our opinion, α-PiHP and its metabolite concentration database can be helpful in the interpretation of fatal cases.

RELEVANCE OF THE USE OF POSTMORTEM BIOMATERIAL OF DOMESTICATED YAK (BOS GRUNNIENS) TO OBTAIN STEM CELLS FROM BONE MARROW

The development of new technology methods for cryopreservation of animal cells has contributed to the introduction of stem cell banks for clinical use, including transplantation and regenerative veterinary medicine, and their further use to avoid problems of donors’ shortage. The research aims to determine the possibility of using the bone marrow of a domesticated yak as a source of stem cells after slaughter. Moreover, we should determine the suitability of post-mortem biological material for obtaining stem cells based on the index of proliferation and viability of cultured cells. Therefore, we used the following materials and methods: bone marrow, obtained from the femur of a domesticated yak in the post-slaughter period. Bone marrow samples were taken in compliance with the rules of asepsis in a sterile test tube. Thus, we added 0.25% trypsin solution to the biomass (the ratio of bone marrow to the solution is 10:1) and placed it in a refrigerator (t +40 °C) for 24 hours for enzymatic disaggregation. Furthermore, we carried out the culturing of the obtained cells in a CO2 incubator according to the standard method with passivation after the formation of a monolayer by 90%–100%. When culturing a suspension of cells obtained from post-slaughter bone marrow material, we noted the appearance of cell colonies six days after sowing. Periodic passivation of stem cells contributed to an increase in the biomass of actively proliferating cells. In addition, we found that stem cells isolated from post-slaughter bone marrow material of domesticated yak have significant proliferative potential, as evidenced by proliferation indices in the range from the first to the third passages and high cell viability. Thus, one can use the obtained post-mortem material in the form of the bone marrow of a domesticated yak as an additional source of stem cells. This post-mortem biological material is suitable for the isolation of stem cells 72 hours after the slaughter of an animal, which opens up the possibility of its transportation over long distances.&#x0D; The scientific novelty of the research is in the context of the international Convention on Biological Diversity, which means the variability of living organisms from all sources, including ecosystems and ecological complexes they are a part of. Within the strategy and action plan for the conservation of biodiversity of the Russian Federation, the ecosystem service of livestock production is significant for preserving the traditional way of life of the indigenous peoples of Siberia and the Russian Far East (it has regional and local significance). This global problem can be solved by (1) developing cell technologies for species not yet involved in it and (2) improving existing ones.

Polymeric Carbon Nitrides for Photoelectrochemical Applications: Ring Opening-Induced Degradation

Active and stable materials that utilize solar radiation for promoting different reactions are critical for emerging technologies. Two of the most common polymeric carbon nitrides were prepared by the thermal polycondensation of melamine. The scope of this work is to investigate possible structural degradation before and after photoelectrochemical testing. The materials were characterized using synchrotron radiation and lab-based techniques, and subsequently degraded photoelectrochemically, followed by post-mortem analysis. Post-mortem investigations reveal: (1) carbon atoms bonded to three nitrogen atoms change into carbon atoms bonded to two nitrogen atoms and (2) the presence of methylene terminals in post-mortem materials. The study concludes that polymeric carbon nitrides are susceptible to photoelectrochemical degradation via ring opening.

Development and Validation of Antibodies Against Sars Cov-2 by Immunohistochemistry to Assess the Pathogenesis of COVID-19 and Its Role in Cytokine Storm Syndrome and Coagulopathy

Introduction: Understand how the SARS-Cov-2 virus uses the ACE2, TMPRSS2, and CD147 receptors to infect human cells. Research and better understand “Cytokine Storm Syndrome” and coagulopathy induced by COVID-19 infection. Objectives: To determine the expression, by means of immunohistochemistry (IHC) in FFPE tissues to evaluate the general pathological events caused by the virus. Materials and Methods: Antibodies from Bio SB Inc were used to analyze post-mortem material from SARS-CoV-2 positive patients by immunohistochemistry. Results: The findings confirmed the presence of viral particles, Cytokine Storm Syndrome inflammation effects, and indicators of coagulopathy. Conclusion: We consider that this study has enough material to provide clinicians and researchers with a detailed picture of the pathogenesis of SARSCoV-2.

ИЗУЧЕНИЕ КИШЕЧНОГО МИКРОБИОМА СИБИРСКОЙ КОСУЛИ

The fauna of Yakutia is rich and diverse, but the epizootic situation of the republic with regard to infectious diseases of wild animals remains poorly studied. There are practically no reports in the literature on the study of the microbiota of wild ungulates of the Arctic and Subarctic. The study of the microbiome of wild animals is not only relevant but also informative research, as the microbiota is a sensitive indicator that responds to many factors of the external and internal environment of the body. The aim is to study the microbiome of wild animals in the Arctic and Subarctic zones of Russia. The article presents the results of the study of the intestinal microbiota of Siberian roe deer. The biological material (thin and thick sections of the intestine (lumen and wall microbiota)) was studied. The generally accepted dilution method was used for quantitative counting of microorganisms. Analysis of the results showed that in the studied Siberian roe deer (43 heads), aerobic bacilli of the genus Bacillus dominate in the normal microbiota (wall and lumen) up to 88–100% (respectively), the main representatives of the intestinal normoflora are significantly inferior to them are: bifidobacteria (61.3–68.1%), lactobacilli (36.3–63.6%), enterococci (59–66%), and lactosepositive escherichia (54.5–59%). Quantitatively, the intestinal microbiocenosis of roe deer showed 135 a comparatively high number of bacilli – up to 104 CFU/g, the number of lactobacilli, enterococci and lactose-positive escherichia was much less, and bifidobacteria were registered in a significantly low amount – up to 101 CFU/g. Examination of material taken from the carcasses of dead roe deer revealed the absence of representatives of normal microflora, except for lactose-positive escherichia, which were found in the wall contents. Microbiological examination of the post-mortem material (parenchymal organs and intestines) revealed potential enteropathogens – lactose-negative escherichia, which, were further identified as Salmonella abortus equi, with high virulence to laboratory animals. The conducted studies showed pronounced intestinal dysbiosis in fallen roe deer, which is characterised by the absence of the main representatives of normal microflora along with the predominance of a large number of potential enteropathogens up to 104 CFU/g. The presence of a highly virulent causative agent of salmonellosis in the intestinal microbiota of roe deer indicates their etiological role in the cause of animal deaths. Thus, summarizing the results of the conducted studies, it can be concluded that aerobic bacilli of the genus Bacillus dominate in the intestinal microbiota of the Siberian roe deer and they have the main protective function against pathogenic microorganisms.

New Synthetic Cannabinoid ADB-BUTINACA-Related Death of a Police Dog.

The popularity of synthetic cannabinoids puts police sniffer dogs at risk of accidental introduction of such substances into the body. The extreme efficacy and potency of many new synthetic cannabinoids are associated with a high risk of serious poisonings and even deaths. The paper presents the toxicological findings in an intoxication of a police dog, in which a new synthetic cannabinoid ADB-BUTINACA was detected and quantified in postmortem materials. The screening analyses were performed by liquid chromatography with tandem mass spectrometry (LC-MS-MS) and liquid chromatography--quadrupole/time-of-flight mass spectrometry (LC-QTOF-MS). LC-MS-MS was also used for quantitative analyses, while LC-QTOF-MS for metabolite identification. Due to unusual matrices, the standard addition method was used for the quantitative determination of ADB-BUTINACA. The determined concentrations of ADB-BUTINACA in blood, lung, stomach, liver and kidney were 8.1 ng/mL, 6.4 ng/g, 1.5 ng/g, 1.8 ng/g and 0.4 ng/g, respectively. Apart from ADB-BUTINACA, the monohydroxylated metabolites and the dihydrodiol metabolite were detected and identified in all analyzed materials, and moreover the product of N-debutylation was found in blood and liver. The described case presents the identification and quantitation of a new synthetic cannabinoid ADB-BUTINACA in postmortem dog specimens. Although the cause of death was acute gastric dilatation, it cannot be ruled out that this process was the result of synthetic cannabinoid inhalation. Due to dogs' sensitivity to cannabinoids, ADB-BUTINACA poisoning cannot be excluded either. The described case suggests that ADB-BUTINACA elicits serious adverse effects in dogs. The article also indicates the dangers to which police dogs coming into contact with extremely potent drugs may be exposed.

EXPRESSION OF THE WATER CHANNEL PROTEIN AQUAPORIN-4 IN THE BRAIN DURING HUMAN LIVER CIRRHOSIS

Hepatic encephalopathy (HE) is a frequent complication of liver cirrhosis, manifesting as a neuropsychiatric syndrome ranging from cognitive deficits to coma. HE pathophysiology is linked to elevated brain ammonia and neuroinflammation [1]. Astrocytes are central brain cells responsible for ammonia detoxification and during acute HE are characterized by severe swelling [1]. Aquaporin-4 (AQP4) is a central protein of astrocytes, predominant water channel in the brain, which undergo alteration in response to hyperammonemia [2]. Studies have evidenced that AQP4 which is enriched in astrocytic perivascular end-feet and responsible for the brain water homeostasis, can be either upregulated or reduced in acute and chronic liver diseases accompanied by hyperammonemia [2]. Despite the controversial findings, it was supposed that AQP4 alteration may play a principal role in cytotoxic and/or vasogenic edema formation occurring during HE [3]. This statement needs further arguments to uncover the mechanisms that control edematous changes during liver cirrhosis in humans. Thus, the present study aimed to study of AQP4 level in 6 brain regions of cirrhotic patients in the course of liver cirrhosis. For this, we examined postmortem material of 90 cirrhotic patients of classes A, B and C according to Child-Pugh classification. Immunohistochemically, using rabbit polyclonal anti-AQP4 (Thermo Scientific, USA), we studied cortex, subcortical white matter, hippocampus, thalamus, striatum and cerebellum. Additionally, data from case histories were analyzed. It was revealed that AQP4+ labeling in all studied regions in control was related to the membranes of perivascular and parenchymal astrocytic processes of individual astrocytes and AQP4 expression appeared to be the highest in the hippocampus and the lowest in the white matter. In cirrhotic groups, AQP4 expression altered in growing manner and correlated with liver cirrhosis aggravation. Increased AQP4 expression was associated with labeling of cell body’s plasmalemmas and increased numbers of positive cells in all studied regions, which caused moderate-to-weak homogenous staining of neuropil. Class A demonstrated increased AQP4 in all studied regions with the highest values in the striatum – 2.62-fold and the least in the cerebellum – 1.66-fold. In class B, AQP4 elevation gained maximal indications of 3.73-fold increase in thalamus, 3.37-fold in the cortex and the least increase in hippocampus – 2.41-fold. The highest increase of AQP4 was observed in the class C. Cortical and thalamic regions showed the most prominent elevation, respectively: 4.25-fold and 4.34-fold. The least AQP4 elevation was related to cerebellum: 2.92-fold. AQP4 expression differed significantly in all pairs of subsequent cirrhotic classes in the white matter, thalamus, striatum and cerebellum. In the cortex and hippocampus, AQP4 levels differed significantly between A vs. B and A vs. C, but not between B vs. C classes.