Detection of prions in matching post-mortem skin and cerebrospinal fluid samples using second-generation real-time quaking-induced conversion assay.

Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases´ diagnostics.

Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles.

Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in ß-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of ß-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs.

Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis.

Different biomarkers are investigated to detect the causes of severe complications in preterm infants. Extracellular vesicles (EVs) are recognized as an important part of cell-to-cell communication, and their increased levels were reported in numerous pathological states. We aimed to increase our knowledge about the incidence of platelet and endothelial EVs in cord blood of preterm newborns using conventional flow cytometry. The presence of platelet (CD36+CD41+), activated platelet (CD41+CD62+), and endothelial (CD31+CD105+) EVs was analyzed. Immune electron microscopy was used to confirm the presence of EVs and the specificity of their labeling. The size of detected extracellular vesicles was in the range 400-2000 nm. The differences in the counts of EVs between the preterm and control group were not significant and no correlation of EVs count with gestation age was recorded. Cord blood plasma samples with free hemoglobin level > 1 mg/mL had more than threefold higher counts of CD36+CD41+ and CD41+CD62+ EVs (p < 0.001), while the count of CD31+CD105+ EVs was only moderately increased (p < 0.05). Further studies utilizing cytometers with improved sensitivity are needed to confirm that the analysis of large platelet and endothelial EVs mirrors the quantitative situation of their whole plasma assemblage.

Detection of Prions in Brain Homogenates and CSF Samples Using a Second-Generation RT-QuIC Assay: A Useful Tool for Retrospective Analysis of Archived Samples.

The possibilities for diagnosing prion diseases have shifted significantly over the last 10 years. The RT-QuIC assay option has been added for neuropsychiatric symptoms, supporting biomarkers and final post-mortem confirmation. Samples of brain homogenates used for final diagnosis, archived for many years, provide the possibility for retrospective studies. We used a second-generation RT-QuIC assay to detect seeding activity in different types of sporadic and genetic prion diseases in archival brain homogenates and post-mortem CSF samples that were 2 to 15 years old. Together, we tested 92 archival brain homogenates: 39 with definite prion disease, 28 with definite other neurological disease, and 25 with no signs of neurological disorders. The sensitivity and specificity of the assay were 97.4% and 100%, respectively. Differences were observed in gCJD E200K, compared to the sporadic CJD group. In 52 post-mortem CSF samples-24 with definite prion disease and 28 controls-we detected the inhibition of seeding reaction due to high protein content. Diluting the samples eliminated such inhibition and led to 95.8% sensitivity and 100% specificity of the assay. In conclusion, we proved the reliability of archived brain homogenates and post-mortem CSF samples for retrospective analysis by RT-QuIC after long-term storage, without changed reactivity.

Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5.

BACKGROUND INFORMATION: Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. RESULTS: Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal ß-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal. CONCLUSIONS: PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. SIGNIFICANCE: Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.

Optimization of the photodynamic inactivation of prions by a phthalocyanine photosensitizer: The crucial involvement of singlet oxygen.

Prion disorders are fatal neurodegenerative diseases caused by the autocatalytic conversion of a natively occurring prion protein (PrPC ) into its misfolded infectious form (PrPTSE ). The proven resistance of PrPTSE to common disinfection procedures increases the risk of prion transmission in medical settings. Herein, we present the effective photodynamic inactivation (PDI) of prions by disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO3 )2 ) utilizing two custom-built red light sources. The treatment eliminates PrPTSE signal in infectious mouse brain homogenate with efficiency that depends on light intensity but has a low effect on the overall protein content. Importantly, singlet oxygen (O2 (1 Δg )) is the only species significantly photogenerated by AlPcOH(SO3 )2 , and it is responsible for the PDI of prions. More intensive light conditions show not only higher O2 (1 Δg ) production but also decreases in AlPcOH(SO3 )2 photostability. Our findings suggest that PDI by AlPcOH(SO3 )2 -generated O2 (1 Δg ) represents a promising approach for prion inactivation that may be useful in future decontamination strategies for delicate medical tools.

An effective "three-in-one" screening assay for testing drug and nanoparticle toxicity in human endothelial cells.

Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7-14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 µg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 µg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented "three-in-one" screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.

Precision in the design of an experimental study deflects the significance of proteinase-activated receptor 2 expression in scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) is suspected to modulate the pathogenesis of various neurodegenerative conditions. We previously described delayed onset of clinical symptoms and prolonged survival of PAR2-deficient mice after intracerebral inoculation with prions. Here we report the results from a refined blinded study that aimed to investigate the effects of PAR2 deletion on scrapie pathogenesis after peripheral infection. This study failed to confirm that PAR2 deficiency impacts on the length of the incubation period, with PAR2-/- and PAR2+/+ littermates developing scrapie at the same time. To clarify the discrepancy between the two observations, we repeated the intracerebral inoculation study while utilizing our refined protocol, which aimed to limit possible sources of experimental bias. The study again failed to confirm the significant effect of PAR2 expression on the course of prion infection. Our report emphasizes and discusses the importance of unbiased experimental design and the selection of proper genetic controls when using genetically altered animal models for prion pathogenesis studies.

Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity.

Quinacrine-the drug based on 9-aminoacridine-failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure because of the transfer of acridine moiety from quinacrine to abundant glutathione. Here, we described the semi-large-scale synthesis of the acridinylated glutathione and the consequences of its formation on biological and biophysical activities. The acridinylated glutathione is one order of magnitude weaker prion protein binder than the parent quinacrine. Moreover, according to log DpH 7.4 , the glutathione conjugate is two orders of magnitude more hydrophilic than quinacrine. Its higher hydrophilicity and higher dsDNA binding potency will significantly decrease its bioavailability in membrane-like environment. The glutathione deactivates quinacrine not only directly but also decreases its bioavailability. Furthermore, the conjugate can spontaneously decompose to practically insoluble acridone, which is precipitated out from the living systems.

Production of mouse embryonic stem cell lines from maturing oocytes by direct conversion of meiosis into mitosis.

ESCs are most commonly derived from embryos originating from oocytes that reached metaphase II. We describe here a novel approach where ESCs with all pluripotency parameters were established from oocytes in which metaphase I was converted, from the cell cycle perspective, directly into metaphase II-like stage without the intervening anaphase to telophase I transition. The resulting embryos initiate development and reach the blastocyst stage from which the ESC lines are then established. Thus, our approach could represent an ethically acceptable method that can exploit oocytes that are typically discarded in in vitro fertilization clinics. Moreover, our results also indicate that the meiotic cell cycle can be converted into mitosis by modulating chromosomal contacts that are typical for meiosis with subsequent licensing of chromatin for DNA replication.

The inability of fully grown germinal vesicle stage oocyte cytoplasm to transcriptionally silence transferred transcribing nuclei.

For somatic cell nuclear transfer cytoplasts from metaphase II, oocytes are exclusively used. However, it is evident that certain reprogramming activities are present in oocytes even at earlier stages of maturation. These activities are, however, only poorly characterised. The main reason for this is that even the intrinsic oocyte processes are insufficiently understood. The mammalian oocyte is a highly specialised cell that harbours many specific characteristics. One of these is its particularly large size when compared to somatic cells. As the oocyte enters the growth phase its volume, as well as the amount of material, increases considerably. Thus, it is clear that the oocyte must possess the machinery to accomplish this incredible material accumulation. When the growth phase is completed, the transcription ceases and the oocyte becomes transcriptionally inactive. In our study, we have used the model system of oocyte fusion (transcribing x non-transcribing germinal vesicle (GV) stage oocytes) as a substitute for a somatic cell nuclear transfer schemes where the somatic cell nucleus would be introduced into a cytoplast obtained from a GV stage oocyte. We wanted to determine if the fully grown GV stage oocyte could induce reprogramming of transcriptionally active transferred nucleus by suppressing this activity. In order to evaluate possible changes in transcriptional properties after nuclear transfer, we also investigated the mechanism of transcriptional silencing taking place when the oocyte reaches its full size as well as the fate of the components namely of the RNA polymerase II (Pol II) transcriptional and splicing machinery. Here, we show that while the Pol II is degraded in fully grown GV stage oocytes and the splicing proteins undergo significant rearrangement, these oocytes are unable to induce similar changes in transcriptionally active nuclei even after a prolonged culture interval.

How to repair the oocyte and zygote?

The supply of human oocytes is very limited. This restricts not only certain assisted reproduction procedures in IVF clinics where recipients wait for oocytes from donors, but also development of some promising approaches, like therapeutic nuclear transfer with subsequent derivation of patient compatible embryonic stem cells. Moreover, in some patients, collected oocytes exhibit certain specific defects, and logically, we can expect that after fertilization, the embryos arising from these defective oocytes may not develop or that their development might eventually be compromised. For this reason, an increased effort to determine how to repair oocytes is evident in the literature. In general, abnormalities (defects) can be detected in different oocyte components, the zona pellucida, cytoplasm, nucleus (chromosomes) and nucleolus. Whereas defects of a nuclear component are impossible (nuclear DNA) or very hard to repair (nucleolus), zona pellucida abnormalities and cytoplasm defects (for example, if containing mutated mitochondrial DNA, mtDNA) can be repaired in some cases with the help of micromanipulation schemes. In the present article, we will briefly outline the current methodological approaches that can be used to repair the oocyte or one-cell stage embryo.

Epigenetic analysis of human spermatozoa after their injection into ovulated mouse oocytes.

BACKGROUND: The epigenetic status of human spermatozoa is difficult to analyse. The method of interspecies fertilization can be used for different purposes. The aim of our work was to adopt this approach for the detailed analysis of epigenetic status of human spermatozoa injected into mouse oocytes. METHODS: Human spermatozoa were injected into ovulated mouse oocytes. When both parental pronuclei were formed, the zygotes were fixed and labeled with antibodies against histones methylated or acetylated at different positions (residues). RESULTS: Our results show that human spermatozoa injected into mouse oocytes fully respond to oocyte cytoplasmic factors and form analysable pronuclei. The labeling of zygotes showed that as in other species, the paternal chromatin is extensively epigenetically remodeled. CONCLUSIONS: The interspecies ICSI may be a powerful tool for the analysis of sperm epigenetic status even with a very low number of spermatozoa available. This analysis could be used as an additional approach for the assessment of certain forms of human infertility, as well as for testing the normality of male gametes obtained from embryonic stem cells.