A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Comparison of the Variability of Small Extracellular Vesicles Derived from Human Liver Cancer Tissues and Cultured from the Tissue Explants Based on a Simple Enrichment Method

A potential use of small extracellular vesicles (sEVs) for diagnostic and therapeutic purposes has recently generated a great interest. sEVs, when purified directly from various tissues with proper procedures, can reflect the physiological and pathological state of the organism. However, the quality of sEV is affected by many factors during isolation, including separation of sEV from cell and tissues debris, the use of enzymes for tissue digestion, and the storage state of tissues. In the present study, we established an assay for the isolation and purification of liver cancer tissues-derived sEVs (tdsEVs) and cultured explants-derived sEVs (cedsEVs) by comparing the quality of sEVs derived from different concentration of digestion enzyme and incubation time. The nano-flow cytometry (NanoFCM) showed that the isolated tdsEVs by our method are purer than those obtained from differential ultracentrifugation. Our study thus establishes a simple and effective approach for isolation of high-quality sEVs that can be used for analysis of their constituents. Graphical abstract

Toxoplasma gondii infection in slaughtered domestic ruminants in Northwest Ethiopia: occurrence, bioassay and virulence assessment

Purpose This study investigated the seropositivity, isolation and virulence of Toxoplasma gondii in slaughtered domestic ruminants in Gondar city, Northwest Ethiopia. Methods Three hundred thirty-five blood samples (135 sheep, 50 goats and 150 cattle) were collected from slaughterhouses. Antibodies against T. gondii were assayed using a commercial Toxo-Latex agglutination test. Tissue digestion was also conducted on 39 heart muscles of seropositive animals using the pepsin enzyme, and isolation of viable T. gondii, from seropositive ruminants, was also performed in white albino mice. Results The overall occurrence of T. gondii infection was 55.8%. The occurrence of T. gondii antibodies in cattle, goats and sheep was 59.3%, 58%, and 51.1%, respectively. Toxoplasma gondii antibodies prevalence was significantly higher in females (χ2 = 4.55, p < 0.033) and adults of sheep (χ2 = 7.57, p < 0.006). Similarly, in cattle, old groups (χ2 = 7.81, p < 0.005) and cross-breeds (χ2 = 6.30, p < 0.012) have presented association with presence of T. gondii antibodies. However, in goats, no association was observed either with sex or age groups. In bioassayed mice, the overall viable T. gondii isolates were 38.5% and the parasites were isolated from samples of sheep (8/16), cattle (3/14) and goats (4/9), and most of these isolates (87.2%) were avirulent. Conclusion The high occurrence of T. gondii antibodies and a high proportion of viable T. gondii observed in this study indicated the prevalent nature of the parasite and its zoonotic importance in the study areas, where slaughtered domestic ruminants serve as an important human protein source. Education of the public about routes of T. gondii transmission and control methods is imperative to prevent T. gondii transmission.

Image-Based Method to Quantify Decellularization of Tissue Sections

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Toxoplasma gondii infection in slaughtered domestic ruminants in Northwest Ethiopia: seropositivity, bioassay and virulence assessment

This study investigated the seropositivity, isolation and virulence of Toxoplasma gondii in slaughtered domestic ruminants in Gondar city, Northwest Ethiopia. Three hundred thirty-five blood samples (135 sheep, 50 goats and 150 cattle) were collected from slaughterhouses. Antibodies against T. gondii were assayed using a commercial Toxo-Latex agglutination test. Tissue digestion was also conducted on 39 heart muscles of seropositive animals using the pepsin enzyme. The isolation of viable T. gondii from seropositive ruminants was also performed in white albino mice. The overall seroprevalence of T. gondii infection was found to be 55.8%. The species-wise prevalence of T. gondii seropositivity in cattle, goats and sheep was 59.3%, 58%, and 51.1%, respectively. From observed risk factors, sex (p < 0.033) and age of the sheep (p < 0.006) showed a significant association with T. gondii seropositivity. Similarly, in cattle, age (p < 0.005) and breed (p < 0.012) showed a statistically significant association with seropositivity of anti-T. gondii antibodies. In bioassayed mice, the overall viable T. gondii isolates were 38.5% and most of these isolates (87.18%) were avirulent. In conclusion, the high prevalence of T. gondii antibody and a high proportion of viable T. gondii observed in this study indicated the prevalent nature of the parasite and its zoonotic importance in the study areas where slaughtered domestic ruminants serve as an important human protein source. Education of the public about routes of T. gondii transmission and control methods is imperative to prevent T. gondii transmission.

Toxoplasma gondii infection in slaughtered domestic ruminants in Northwest Ethiopia: seropositivity, bioassay and virulence assessment

This study investigated the seropositivity, isolation and virulence of Toxoplasma gondii in slaughtered domestic ruminants in Gondar city, Northwest Ethiopia. Three hundred thirty-five blood samples (135 sheep, 50 goats and 150 cattle) were collected from slaughterhouses. Antibodies against T. gondii were assayed using a commercial Toxo-Latex agglutination test. Tissue digestion was also conducted on 39 heart muscles of seropositive animals using the pepsin enzyme. The isolation of viable T. gondii from seropositive ruminants was also performed in white albino mice. The overall seroprevalence of T. gondii infection was found to be 55.8%. The species-wise prevalence of T. gondii seropositivity in cattle, goats and sheep was 59.3%, 58%, and 51.1%, respectively. From observed risk factors, sex (p < 0.033) and age of the sheep (p < 0.006) showed a significant association with T. gondii seropositivity. Similarly, in cattle, age (p < 0.005) and breed (p < 0.012) showed a statistically significant association with seropositivity of anti-T. gondii antibodies. In bioassayed mice, the overall viable T. gondii isolates were 38.5% and most of these isolates (87.18%) were avirulent. In conclusion, the high prevalence of T. gondii antibody and a high proportion of viable T. gondii observed in this study indicated the prevalent nature of the parasite and its zoonotic importance in the study areas where slaughtered domestic ruminants serve as an important human protein source. Education of the public about routes of T. gondii transmission and control methods is imperative to prevent T. gondii transmission.

Microfluidic platform accelerates tissue processing into single cells for molecular analysis and primary culture models

Tissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues.

A Method for Rapid Flow-cytometric Isolation of Endothelial Nuclei and RNA from Archived Frozen Brain Tissue

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

A Tissue Digestion Protocol for Measuring Sarcoptes scabiei (Astigmata: Sarcoptidae) Density in Skin Biopsies

Sarcoptic mange is a parasitic skin disease caused by the burrowing mite Sarcoptes scabiei that affects a diversity of mammals, including humans, worldwide. In North America, the most commonly affected wildlife includes wild canids, such as coyotes and red foxes, and more recently American black bears in the Mid-Atlantic and Northeast United States. Currently, surveillance for sarcoptic mange in wildlife is syndromic, relying on detection of clinical signs and lesions, such as alopecia and crusting of skin. When possible, skin scrapes are used to identify the causative mite. While skin scrapes are a valuable diagnostic tool to identify mites, this approach has significant limitations when used for quantification of mite burden. To further investigate mite burden in cases of sarcoptic mange, 6-mm punch biopsies were collected from affected skin of red foxes (Vulpes vulpes Linnaeus [Carnivora: Canidae]), a species historically affected by sarcoptic mange, frequently with high mite burdens and severe skin disease, and validated on skin tissue from mange-affected American black bears (Ursus americanus Pallas [Carnivora: Ursidae]) and coyotes (Canis latrans Say [Carnivora: Canidae]). Biopsies were digested by incubating the tissue in potassium hydroxide (KOH) at 55°C. The greatest tissue clearance and lowest mite degradation resulted after 12 h of tissue digestion. The purpose of this manuscript is to describe a methodology for host tissue digestion and mite quantification in cases of sarcoptic mange. This method will provide a valuable surveillance and research tool to better understand sarcoptic mange in wild and domestic animals, with applications to a diversity of other ectoparasitic diseases.