High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue.

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

Dual spatially resolved transcriptomics for human host-pathogen colocalization studies in FFPE tissue sections.

Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host-pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples.

A latent cardiomyocyte regeneration potential in human heart disease.

Cardiomyocytes in the adult human heart show a regenerative capacity, with an annual renewal rate around 0.5%. Whether this regenerative capacity of human cardiomyocytes is employed in heart failure has been controversial. Using retrospective 14C birth dating we analyzed cardiomyocyte renewal in patients with end-stage heart failure. We show that cardiomyocyte generation is minimal in end-stage heart failure patients at rates 18-50 times lower compared to the healthy heart. However, patients receiving left ventricle support device therapy, who showed significant functional and structural cardiac improvement, had a >6-fold increase in cardiomyocyte renewal relative to the healthy heart. Our findings reveal a substantial cardiomyocyte regeneration potential in human heart disease, which could be exploited therapeutically.

FiNuTyper: Design and validation of an automated deep learning-based platform for simultaneous fiber and nucleus type analysis in human skeletal muscle.

AIM: While manual quantification is still considered the gold standard for skeletal muscle histological analysis, it is time-consuming and prone to investigator bias. To address this challenge, we assembled an automated image analysis pipeline, FiNuTyper (Fiber and Nucleus Typer). METHODS: We integrated recently developed deep learning-based image segmentation methods, optimized for unbiased evaluation of fresh and postmortem human skeletal muscle, and utilized SERCA1 and SERCA2 as type-specific myonucleus and myofiber markers after validating them against the traditional use of MyHC isoforms. RESULTS: Parameters including cross-sectional area, myonuclei per fiber, myonuclear domain, central myonuclei per fiber, and grouped myofiber ratio were determined in a fiber-type-specific manner, revealing that a large degree of sex- and muscle-related heterogeneity could be detected using the pipeline. Our platform was also tested on pathological muscle tissue (ALS and IBM) and adapted for the detection of other resident cell types (leucocytes, satellite cells, capillary endothelium). CONCLUSION: In summary, we present an automated image analysis tool for the simultaneous quantification of myofiber and myonuclear types, to characterize the composition and structure of healthy and diseased human skeletal muscle.

Bioprotective role of platelet-derived microvesicles in hypothermia: Insight into the differential characteristics of peripheral and splenic platelets.

BACKGROUND: Most platelets are present in peripheral blood, but some are stored in the spleen. Because the tissue environments of peripheral blood vessels and the spleen are quite distinct, the properties of platelets present in each may also differ. However, no studies have addressed this difference. We previously reported that hypothermia activates splenic platelets, but not peripheral blood platelets, whose biological significance remains unknown. In this study, we focused on platelet-derived microvesicles (PDMVs) and analyzed their biological significance connected to intrasplenic platelet activation during hypothermia. METHODS: C57Bl/6 mice were placed in an environment of -20 °C, and their rectal temperature was decreased to 15 °C to model hypothermia. Platelets and skeletal muscle tissue were collected and analyzed for their interactions. RESULTS: Transcriptomic changes between splenic and peripheral platelets were greater in hypothermic mice than in normal mice. Electron microscopy and real-time RT-PCR analysis revealed that platelets activated in the spleen by hypothermia internalized transcripts, encoding tissue repairing proteins, into PDMVs and released them into the plasma. Plasma microvesicles from hypothermic mice promoted wound healing in the mouse myoblast cell line C2C12. Skeletal muscles in hypothermic mice were damaged but recovered within 24 h after rewarming. However, splenectomy delayed recovery from skeletal muscle injury after the mice were rewarmed. CONCLUSIONS: These results indicate that PDMVs released from activated platelets in the spleen play an important role in the repair of skeletal muscle damaged by hypothermia.

A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Prostate cancer disease recurrence after radical prostatectomy is associated with HLA type and local cytomegalovirus immunity.

Prostate cancer is a heterogeneous disease with a need for new prognostic biomarkers. Human leukocyte antigen (HLA) genes are highly polymorphic genes central to antigen presentation to T-cells. Two alleles, HLA-A*02:01 and HLA-A*24:02, have been associated with prognosis in patients diagnosed with de novo metastatic prostate cancer. We leveraged the next-generation sequenced cohorts CPC-GENE and TCGA-PRAD to examine HLA alleles, antiviral T-cell receptors and prostate cancer disease recurrence after prostatectomy. Carrying HLA-A*02:01 (111/229; 48% of patients) was independently associated with disease recurrence in patients with low-intermediate risk prostate cancer. HLA-A*11 (carried by 42/441; 10% of patients) was independently associated with rapid disease recurrence in patients with high-risk prostate cancer. Moreover, HLA-A*02:01 carriers in which anti-cytomegalovirus T-cell receptors (CMV-TCR) were identified in tumors (13/144; 10% of all patients in the cohort) had a higher risk of disease recurrence than CMV-TCR-negative patients. These findings suggest that HLA-type and CMV immunity may be valuable biomarkers for prostate cancer progression.

Diploid hepatocytes drive physiological liver renewal in adult humans.

Physiological liver cell replacement is central to maintaining the organ's high metabolic activity, although its characteristics are difficult to study in humans. Using retrospective radiocarbon (14C) birth dating of cells, we report that human hepatocytes show continuous and lifelong turnover, allowing the liver to remain a young organ (average age <3 years). Hepatocyte renewal is highly dependent on the ploidy level. Diploid hepatocytes show more than 7-fold higher annual birth rates than polyploid hepatocytes. These observations support the view that physiological liver cell renewal in humans is mainly dependent on diploid hepatocytes, whereas polyploid cells are compromised in their ability to divide. Moreover, cellular transitions between diploid and polyploid hepatocytes are limited under homeostatic conditions. With these findings, we present an integrated model of homeostatic liver cell generation in humans that provides fundamental insights into liver cell turnover dynamics.

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation.

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP−5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain.

Hypoxia-induced nuclear translocation of ß-catenin in the healing process of frostbite.

Frostbite occurs when the skin is exposed to localized low temperatures. The main causes of frostbite are thought to be direct cell injury due to freezing of cells and tissue ischemia due to abnormal blood circulation. However, the molecular mechanism of frostbite has not been elucidated. This study aims to explain the molecular dynamics of frostbite using a mouse frostbite model and keratinocyte cell culture. Comprehensive gene expression analysis performed on mouse skin samples revealed that ß-catenin signaling is activated by frostbite. Immunohistochemistry showed nuclear translocation of ß-catenin in the skin of frostbite model mice that was not observed in mice subjected to a mechanical skin damage model induced by tape stripping. Tissue hypoxia, as detected by pimonidazole staining, coexisted with nuclear expression of ß-catenin. In keratinocyte cell cultures, nuclear translocation of ß-catenin was induced by hypoxia, but not by low temperature. Hypoxia induced epithelial-mesenchymal transition - an important biological event in the healing process of skin - and in vitro wound-healing activity, both of which were suppressed by ß-catenin inhibition. Our results suggest that during frostbite, impaired blood flow causes hypoxia, which in turn activates ß-catenin that promotes keratinocyte motility and tissue repair.

Hypothermia causes platelet activation in the human spleen.

BACKGROUND: Accidental hypothermia results in various dysfunctions in the human body. Additionally, coagulation disorder can lead to a life-threatening condition. We previously demonstrated that platelets stored in the spleen were activated and thus triggered coagulation disorder in a mouse model of hypothermia. In the present study, we wanted to investigate if this phenomenon in mice also occurs in humans as a reaction to hypothermia. METHODS: We analyzed splenic tissue collected from 22 deceased subjects who have died from hypothermia. These samples were compared with 22 control cases not exposed to cold environment. We performed immunohistochemical staining for CD61 (a marker of all platelets) and CD62P (a marker of activated platelets). We also evaluated the morphology of platelets in the spleen with scanning electron microscopy. RESULTS: Immunohistochemical analysis revealed no significant changes in the amounts of CD61-positive platelets between the hypothermia and control cases. However, the hypothermia cases contained abundant CD62P-positive platelets compared with those of the control cases. Immunohistochemical analysis also revealed that the activated platelets formed aggregates and adhered to splenic sinusoidal endothelial cells in the hypothermia cases. However, we observed no significant fibrin formation around the activated platelets. CONCLUSIONS: Hypothermia resulted in splenic platelet activation, which may be used as a postmortem marker of hypothermia. The release of activated platelets from the spleen into to circulation upon rewarming may promote coagulation disturbances.

A Rapid Method for Postmortem Vitreous Chemistry-Deadside Analysis.

Vitreous fluid is commonly collected for toxicological analysis during forensic postmortem investigations. Vitreous fluid is also often analyzed for potassium, sodium, chloride and glucose for estimation of time since death, and for the evaluation of electrolyte imbalances and hyperglycemia, respectively. Obtaining such results in the early phase of a death investigation is desirable both in regard to assisting the police and in the decision-making prior to the autopsy. We analyzed vitreous fluid with blood gas instruments to evaluate/examine the possible impact of different sampling and pre-analytical treatment. We found that samples from the right and left eye, the center of the eye as well as whole vitreous samples gave similar results. We also found imprecision to be very low and that centrifugation and dilution were not necessary when analyzing vitreous samples with blood gas instruments. Similar results were obtained when analyzing the same samples with a regular multi-analysis instrument, but we found that such instruments could require dilution of samples with high viscosity, and that such dilution might impact measurement accuracy. In conclusion, using a blood gas instrument, the analysis of postmortem vitreous fluid for electrolytes and glucose without sample pretreatment produces rapid and reliable results.

A roadmap to the safe practice of forensic medicine in the COVID-19 pandemic.

The COVID-19 pandemic has forced forensic practitioners to consider how we perform our normal duties, especially when those duties involve humans. The potential for contracting the virus from working in close contact with living sufferers is high, and we have yet to fully determine the risk of infection from the deceased. In an attempt to support the community, the Journal of Forensic & Legal Medicine has drawn together three articles which underline the importance of continued forensic medical practice during the pandemic and highlight some factors to consider in a Roadmap towards safe practice. Our Roadmap has intentionally taken an international perspective and supports other work we have published in the Journal on our collective response to the COVID-19 crisis.

Rewarming from accidental hypothermia enhances whole blood clotting properties in a murine model.

BACKGROUND: Hypothermia triggers coagulation, which can lead to the development of a life-threatening condition. We previously reported that hypothermia induces platelet activation in the spleen, resulting in microthrombosis after rewarming. However, the changes in whole blood clotting properties that occur remain unclear. Using thromboelastography, we investigated blood clotting activity and the effects of rewarming in a murine model of hypothermia. METHODS: C57Bl/6 mice were exposed to an ambient temperature of -20 °C under general anesthesia until their rectal temperature decreased to 15 °C. One group of mice was kept at 4 °C for 2 h and then euthanized. Another group was rewarmed, kept in normal conditions for 24 h, and then euthanized. Tissue and citrated whole blood samples were obtained from the mice for histopathological analysis, flow cytometry, and thromboelastography. RESULTS: Hypothermia induced the activation of platelets in the spleen; however, rewarming significantly reduced the number of activated platelets in the spleen while their numbers significantly increased in peripheral blood. In hypothermic mice not subjected to rewarming, no increase in activated platelets was observed in peripheral blood. Thromboelastography analysis showed that whole blood samples from the rewarmed mice displayed an enhanced clotting strength. CONCLUSIONS: Rewarming from hypothermia enhances whole blood coagulation activity accompanied by an increase in the number of active platelets in peripheral blood. This phenomenon may lead to formation of microthrombi and thrombotic disorders.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells.

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture. Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis. Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature. Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

Metformin - Postmortem fatal and non-fatal reference concentrations in femoral blood and risk factors associated with fatal intoxications.

BACKGROUND & OBJECTIVES: To improve the interpretation of fatal intoxications by establishing fatal and non-fatal reference concentrations of metformin in postmortem femoral blood and to further evaluate risk factors associated with fatal metformin intoxication. METHODS: All forensic autopsies in Sweden where metformin was detected in femoral blood 2011-2016 were identified in the National Board of Forensic Medicine databases (NFMD). The cases were classified as single substance intoxications, A (n = 22), multiple substance intoxications, B (N = 7) and postmortem controls, C (N = 13). The control group consisted of cases where metformin was detected, but the cause of death excluded the incapacitation by metformin or other substances. Strict inclusion criteria were used, and all postmortem cases were assessed by two independent reviewers. All other cases where the inclusion criteria of groups A-C where not met formed group O (N = 78). The forensic findings logged in the NFMD where linked to national registers whereby information on comorbidities, dispensed drugs and clinical data could be obtained. RESULTS: The mean age was 66 ± 10 years in the total study population and did not differ between the groups. The proportion of men was 64% in group A, 71% in B, 77% in C and 74% in group O. The median values of metformin in group A (48.5 µg/g; range 13.0-210 µg/g) and B (21.0 µg/g; range 4.40-95.0 µg/g) were significantly (p < 0.001 and p = 0.015 respectively) higher than those of the control group C (2.30 µg/g ; range 0.70-21.0 µg/g). The median concentration of metformin in group A and B was also significantly higher than in group O (4.60 µg/g; range 0.64-54.0 µg/g) (p < 0.001 and p = 0.040 respectively). The results suggest that intoxication with metformin as a cause of death should be considered when the postmortem femoral blood level exceeds about 10 µg/g, although higher levels may be seen in postmortem in cases without incapacitation. The metformin intoxication was confirmed to be intentional in 23% (n = 5) of the single intoxications. Underlying factors identified as important for the remaining fatal metformin intoxications included living alone, any contraindication for the use of metformin, known alcohol abuse and a history of stroke or cardiovascular disease. CONCLUSIONS: The reported post mortem femoral blood concentrations of metformin can hopefully contribute to a better interpretation of results in suspected poisonings and obscure cases. Living in a single household, history of cardiovascular disease and contraindications, predominantly alcohol abuse, were associated with fatal metformin intoxication.

NF-κB-Associated Pain-Related Neuropeptide Expression in Patients with Degenerative Disc Disease.

The role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) has been highlighted in mechanisms underlying inflammatory and neuropathic pain processes. The present study was designed to investigate whether NF-κB signaling is associated with pain-related neuropeptide expression in patients with chronic back pain related to degenerative disc disease (DDD). Intervertebral disc (IVD) tissues were collected from forty DDD patients undergoing disc replacement or fusion surgery, and from eighteen postmortem (PM) control subjects. RELA, NFKB1, CGRP, TAC1, TRPV1, and MMP-3 gene expression were analyzed by RT-qPCR, while NF-κB subunit RelA and NF-κB1⁻DNA binding in nuclear extracts and calcitonin gene related peptide (CGRP), substance P (SP), and transient receptor potential, subfamily V, member 1 (TRPV1) protein levels in cytosolic extracts of tissues were assessed by enzyme-linked immunosorbent assay (ELISA). An upregulated NF-κB1⁻DNA binding, and higher CGRP and TRPV1 protein levels were observed in DDD patients compared to PM controls. In DDD patients, NF-κB1⁻DNA binding was positively correlated with nuclear RelA levels. Moreover, NF-κB1⁻DNA binding was positively associated with TRPV1 and MMP-3 gene and SP and TRPV1 protein expression in DDD patients. Our results indicate that the expression of SP and TRPV1 in IVD tissues was associated with NF-κB activation. Moreover, NF-κB may be involved in the generation or maintenance of peripheral pain mechanisms by the regulation of pain-related neuropeptide expression in DDD patients.

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease.

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

Hippocampal granule cell loss in human chronic alcohol abusers.

Chronic alcohol abuse causes cognitive impairments associated with neurodegeneration and volume loss in the human hippocampus. Here, we hypothesize that alcohol reduces the number of granule cells in the human dentate gyrus and consequently contribute to the observed volume loss. Hippocampal samples were isolated from deceased donors with a history of chronic alcohol abuse and from controls with no alcohol overconsumption. From each case, a sample from the mid-portion of hippocampus was sectioned, immunostained for the neuronal nuclear marker NeuN, and counter stained with hematoxylin. Granule cell number and volume of granular cell layer in the dentate gyrus were estimated using stereology. We found a substantial reduction in granule cell number and also a significantly reduced volume of the granular cell layer of chronic alcohol abusers as compared to controls. In controls there was a slight age-related decline in the number of granule cells and volume of granular cell layer in line with previous studies. This was not observed among the alcoholics, possibly due to a larger impact of alcohol abuse than age on the degenerative changes in the dentate gyrus. Loss of neurons in the alcoholic group could either be explained by an increase of cell death or a reduced number of new cells added to the granular cell layer. However, there is no firm evidence for an increased neuronal death by chronic alcohol exposure, whereas a growing body of experimental data indicates that neurogenesis is impaired by alcohol. In a recent study, we reported that alcoholics show a reduced number of stem/progenitor cells and immature neurons in the dentate gyrus, hence that alcohol negatively affects hippocampal neurogenesis. The present results further suggest that such impairment of neurogenesis by chronic alcohol abuse also results in a net loss of granule cells in the dentate gyrus of hippocampus.