On predatory fungi feeding on free-living amoebae harbouring yeast-like endoparasites.

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium.

Deciphering the Emulsification Process to Create an Albumin-Perfluorocarbon-(o/w) Nanoemulsion with High Shelf Life and Bioresistivity.

This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization. High-pressure homogenization is simple but incorporates complex physical reactions, with many factors influencing the formation of PFC droplets and their coating. This work describes and interprets the impact of albumin concentration, homogenization pressure, and repeated microfluidizer passages on PFC-droplet formation; its influence on storage stability; and the overcoming of obstacles in preparing stable nanoemulsions. The applied methods comprise dynamic light scattering, static light scattering, cryo- and non-cryo-scanning and transmission electron microscopies, nuclear magnetic resonance spectroscopy, light microscopy, amperometric oxygen measurements, and biochemical methods. The use of this wide range of methods provided a sufficiently comprehensive picture of this polydisperse emulsion. Optimization of PFC-droplet formation by means of temperature and pressure gradients results in an emulsion with improved storage stability (tested up to 5 months) that possibly qualifies for clinical applications. Adaptations in the manufacturing process strikingly changed the physical properties of the emulsion but did not affect its oxygen capacity.

Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.

Cisplatin alters the function and expression of N-type voltage-gated calcium channels in the absence of morphological damage of sensory neurons.

Platinum-based chemotherapeutic agents, such as cisplatin, are still frequently used for treating various types of cancer. Besides its high effectiveness, cisplatin has several serious side effects. One of the most common side effects is dorsal root ganglion (DRG) neurotoxicity. However, the mechanisms underlying this neurotoxicity are still unclear and controversially discussed. Cisplatin-mediated modulation of voltage-gated calcium channels (VGCCs) in the DRG neurons has been shown to alter intracellular calcium homeostasis, a process critical for the induction of neurotoxicity. Using the whole-cell patch-clamp technique, immunostaining, behavioural experiments and electron microscopy (EM) of rat DRGs, we here demonstrate that cisplatin-induced neurotoxicity is due to functional alteration of VGCC, but not due to morphological damage. In vitro application of cisplatin (0.5 µM) increased N-type VGCC currents ( ICa(V)) in small DRG neurons. Repetitive in vivo administration of cisplatin (1.5 mg/kg, cumulative 12 mg/kg) increased the protein level of N-type VGCC over 26 days, with the protein level being increased for at least 14 days after the final cisplatin administration. Behavioural studies revealed that N-type VGCCs are crucial for inducing symptoms of cisplatin-related neuropathic pain, such as thermal and mechanical hyperalgesia. EM and histology showed no evidence of any structural damage, apoptosis or necrosis in DRG cells after cisplatin exposure for 26 days. Furthermore, no nuclear DNA damage in sensory neurons was observed. Here, we provide evidence for a mainly functionally driven induction of neuropathic pain by cisplatin.

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice.

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

Identification of compounds that selectively target highly chemotherapy refractory neuroblastoma cancer stem cells.

Relapse of cancer months or years after an apparently successful therapy is probably caused by cancer stem cells (CSCs) due to their intrinsic features like dormant periods, radiorefraction, and acquired multidrug resistance (MDR) phenotypes, among other mechanisms of cellular drug evasiveness. Thus, the lack of currently efficacious interventions remains a major problem in the treatment of malignancies, together with the inability of existing drugs to destroy specifically CSCs. Neuroblastomas per se are highly chemotherapy-refractory extracranial tumors in infants with very low survival rates. So far, no effective cytostatics against this kind of tumors are clinically available. Therefore, we have put much effort into the development of agents to efficiently combat this malignancy. For this purpose, we tested several compounds isolated from Cuban propolis on induced CSCs (iCSC) derived from LAN-1 neuroblastoma cells which expressed several characteristics of tumor-initiating cells both in in-vitro and in-vivo models. Some small molecules such as flavonoids and polycyclic polyprenylated acylphloroglucinols (PPAP) were isolated using successive RT-HPLC cycles and identified employing mass spectrometry and NMR spectroscopic techniques. Their cytotoxicity was first screened in sensitive cell systems by MTT proliferation assays and afterwards studied in less sensitive neuroblastoma iCSC models. We found several compounds with considerable anti-iCSC activity, most of them belonging to the PPAP class. The majority of the compounds act in a pleiotropic manner on the molecular biology of tumors although their specific targets remain unclear. Nevertheless, two substances, one of them a flavonoid, induced a strong disruption of tubulin polymerization. In addition, an unknown compound strongly inhibited replicative enzymes like toposimerases I/II and DNA polymerase. Here, we report for the first time cytotoxic activities of small molecules isolated from Caribbean propolis which could be promising therapeutics or lead structures against therapy-refractory neuroblastoma entities. *Contributed equally.

CEACAM1: a key regulator of vascular permeability.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is an immunoglobulin-like cell surface co-receptor expressed on epithelial, hematopoietic and endothelial cells. CEACAM1 functions as an adhesion molecule, mainly binding to itself or other members of the CEA family. We and others have previously shown that CEACAM1 is crucial for in vivo vascular integrity during ischemic neo-vascularization. Here, we have deciphered the roles of CEACAM1 in normal and pathological vascularization. We have found that Ceacam1-/- mice exhibit a significant increase in basal vascular permeability related to increased basal Akt and endothelial nitric oxide synthase (eNOS) activation in primary murine lung endothelial cells (MLECs). Moreover, CEACAM1 deletion in MLECs inhibits VEGF-mediated nitric oxide (NO) production, consistent with defective VEGF-dependent in vivo permeability in Ceacam1-/- mice. In addition, Ceacam1-null mice exhibit increased permeability of tumor vasculature. Finally, we demonstrate that CEACAM1 is tyrosine-phosphorylated upon VEGF treatment in a SHP-1- and Src-dependent manner, and that the key residues of the long cytoplasmic domain of CEACAM1 are crucial for CEACAM1 phosphorylation and NO production. This data represents the first report, to our knowledge, of a functional link between CEACAM1 and the VEGFR2/Akt/eNOS-mediated vascular permeability pathway.

Inhibition of hyaluronan synthesis accelerates murine atherosclerosis: novel insights into the role of hyaluronan synthesis.

BACKGROUND: Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. METHODS AND RESULTS: Four-week-old apolipoprotein E-deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. CONCLUSIONS: The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.

Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly.

Human spermatozoal RNA features special characteristics such as a significantly reduced quantity within spermatozoa compared with somatic cells is described as being devoid of ribosomal RNAs and is difficult to isolate due to a massive excess of genomic DNA in the lysates. Using a novel two-round column-based protocol for human ejaculates delivering highly purified spermatozoal RNA, we uncovered a heterogeneous, but specific banding pattern in microelectrophoresis with 28S ribosomal RNA being indicative for the amount of round cell contamination. Ejaculates with different round cell quantities and density-purified spermatozoa revealed that 18S rRNA but not 28S rRNA is inherent to a pure spermatozoal fraction. Transmission electron microscopy showed monoribosomes and polyribosomes in spermatozoal cytoplasm, while immunohistochemical results suggest the presence of proteins from small and large ribosomal subunits in retained spermatozoal cytoplasm irrespective of 28S rRNA absence.

Thyroid Hormone Deficiency Modifies Hepatic Lipid Droplet Morphology and Molecular Properties in Lithogenic-Diet Supplemented Mice.

OBJECTIVE: Thyroid hormones have been associated with a hepatic lipid lowering effect and thyroid function has been shown to play a substantial role in development of non-alcoholic fatty liver disease. Hepatic lipid droplets differ in the number, size and molecular properties depending on metabolic state or pathological condition. However, in how far thyroid hormone deficiency affects hepatic lipid droplet morphology and molecular properties is still poorly understood. Therefore, we performed a study in mice using a lithogenic diet model of steatohepatitis and modulated the thyroid hormone status. METHODS: Male and female three months old C57BL/6 mice were divided into a euthyroid (control), a lithogenic (litho) and a lithogenic+thyroid hormone deficient (litho+hypo) group and treated for six weeks. Hepatic transmission electron microscopy and gene expression analysis of lipid-droplet associated proteins were performed. RESULTS: Increased mean diameters of hepatic lipid droplets and a shift towards raised electron-density in lipid droplets was observed under thyroid hormone deficiency. Furthermore thyroid hormone deficiency altered hepatic expression of genes involved in lipophagy and triacylglycerol mobilization. Interestingly, while the impact of thyroid hormone deficiency on lipid droplet morphology seems to be sex-independent, hepatic lipid droplet-associated gene expression differed significantly between both sexes. CONCLUSION: This study demonstrates that thyroid hormone deficiency alters hepatic lipid droplet morphology and hepatic gene expression of lipid droplet-associated proteins in a lithogenic diet mouse model of steatohepatitis.

Hypoxia-inducible factor-2α is crucial for proper brain development.

Sufficient tissue oxygenation is required for regular brain function; thus oxygen supply must be tightly regulated to avoid hypoxia and irreversible cell damage. If hypoxia occurs the transcription factor complex hypoxia-inducible factor (HIF) will accumulate and coordinate adaptation of cells to hypoxia. However, even under atmospheric O2 conditions stabilized HIF-2α protein was found in brains of adult mice. Mice with a neuro-specific knockout of Hif-2α showed a reduction of pyramidal neurons in the retrosplenial cortex (RSC), a brain region responsible for a range of cognitive functions, including memory and navigation. Accordingly, behavioral studies showed disturbed cognitive abilities in these mice. In search of the underlying mechanisms for the specific loss of pyramidal cells in the RSC, we found deficits in migration in neural stem cells from Hif-2α knockout mice due to altered expression patterns of genes highly associated with neuronal migration and positioning.

Characterization of two types of intranuclear hepatocellular inclusions in NAFLD.

Nuclear inclusions (NI) are a common finding in hepatocytes from patients with liver disease especially in diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) but studies examining the shape and content of these inclusions in detail are lacking. In this study we define two distinct types of NI in NAFLD: inclusions bounded by the nuclear membrane, containing degenerative cell organelles and heterolysosomes (type1) and inclusions with deposits of glycogen but without any kind of organelles and delimiting membrane (type2). NI in 77 paraffin-embedded patients of NAFLD including NAFL and non-alcoholic steatohepatitis (NASH) were analyzed. In 4-12% of type1 NI immunopositivity for the autophagy-associated proteins LC3B, ubiquitin, p62/sequestosome1, cathepsin D and cathepsin B were detected with co-localizations of ubiquitin and p62; type2 NI showed no immunoreactivity. Three-dimensional reconstructions of isolated nuclei revealed that NI type1 are completely enclosed within the nucleus, suggesting that NI, although probably derived from cytoplasmic invaginations, are not just simple invaginations. Our study demonstrates two morphologically different types of inclusions in NAFLD, whereby both gained significantly in number in advanced stages. We suggest that the presence of autophagy-associated proteins and degenerated organelles within type1 NI plays a role in disease progression.

Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival.

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival.

Autophagic degradation of lamins facilitates the nuclear egress of herpes simplex virus type 1.

Dendritic cells (DCs) are crucial for the induction of potent antiviral immune responses. In contrast to immature DCs (iDCs), mature DCs (mDCs) are not permissive for infection with herpes simplex virus type 1 (HSV-1). Here, we demonstrate that HSV-1 infection of iDCs and mDCs induces autophagy, which promotes the degradation of lamin A/C, B1, and B2 in iDCs only. This in turn facilitates the nuclear egress of progeny viral capsids and thus the formation of new infectious particles. In contrast, lamin protein levels remain stable in HSV-1-infected mDCs due to an inefficient autophagic flux. Elevated protein levels of KIF1B and KIF2A in mDCs inhibited lamin degradation, likely by hampering autophagosome-lysosome fusion. Therefore, in mDCs, fewer progeny capsids were released from the nuclei into the cytosol, and fewer infectious virions were assembled. We hypothesize that inhibition of autophagic lamin degradation in mDCs represents a very powerful cellular counterstrike to inhibit the production of progeny virus and thus viral spread.

Protection of Oligodendrocytes Through Neuronal Overexpression of the Small GTPase Ras in Hyperoxia-Induced Neonatal Brain Injury.

Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.

Mouse cardiac mitochondria do not separate in subsarcolemmal and interfibrillar subpopulations.

Cardiomyocytes consist of longitudinally oriented myofibril bundles with a misaligned composition caused by the uneven contours of the intercalated discs. The cytoplasmic space harbors the organelles, including mitochondria. This study investigated whether cardiomyocytes contain spatially and ultrastructurally discrete pools of mitochondria that can be separated for structurally and functionally appraisal in (patho)physiology. Transmission electron microscopy disclosed continuous transitions of mitochondria without attributable characteristics from beneath the sarcolemma directly into the barrier-free cytoplasmic space between myofibrils. The various shapes and sizes of mitochondria are formed by myofibril positioning and the space available independent of their localization within the cardiomyocytes. Furthermore, the established enzymatic isolation procedure including proteinase treatment resulted in loss of mitochondrial proteins, as evidenced by immunogold labeling of Connexin43 in situ, a postulated marker for distinguishing mitochondrial subpopulations. Moreover, mitochondrial ATP produced in those mitochondria was not different. These findings preclude a spatial and ultrastructural grading of cardiac mitochondria and their distinct separation and classification in subsarcolemmal and interfibrillar subpopulations.

A Distinct Oncogenerative Multinucleated Cancer Cell Serves as a Source of Stemness and Tumor Heterogeneity.

The effects of anticancer treatments on cell heterogeneity and their proliferative potential play an important role in tumor persistence and metastasis. However, little is known about de-polyploidization, cell fate, and physiologic stemness of the resulting cell populations. Here, we describe a distinctive cell type termed "pregnant" P1 cells found within chemotherapy-refractory ovarian tumors, which generate and gestate daughter generation Gn cells intracytoplasmically. Release of Gn cells occurred by ejection through crevices in the P1 cell membrane by body contractions or using a funiculus-like structure. These events characterized a not yet described mechanism of cell segregation. Maternal P1 cells were principally capable of surviving parturition events and continued to breed and nurture Gn progenies. In addition, P1 cells were competent to horizontally transmit offspring Gn cells into other specific proximal cells, injecting them to receptor R1 cells via cell-cell tunneling. This process represents a new mechanism used by tumor cells to invade surrounding tissues and ensure life cycles. In contrast to the pregnant P1 cells with low expression of stem cell markers despite their physiologic stemness, the first offspring generations of daughter G1 cells expressed high levels of ovarian cancer stem cell markers. Furthermore, both P1 and Gn cells overexpressed multiple human endogenous retroviral envelope proteins. Moreover, programmed death-ligand 1 and the immunosuppressive domain of the retroviral envelope proteins were also overexpressed in P1 cells, suggesting effective protection against the host immune system. Together, our data suggest that P1 oncogenerative cancer cells exhibit a not yet described cell biological mechanism of persistence and transmission of malignant cells in patients with advanced cancers.Significance: P1 oncogenerative cell entities express low levels of CSC markers, which are characteristic of their histological origin. Cancer Res; 78(9); 2318-31. ©2018 AACR.

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.