Podcasts about Immunofluorescence

Blood is a symbol of life, which makes sense given that it plays such an important role in so many body functions, including our immune system. Blood makes up approximately 8% of your normal body weight and unfortunately, cancers of the blood, including lymphoma and leukemia, account for ~10% of all diagnosed cancers in the U.S. each year.  CAR-T cell therapy has emerged as a promising method to engineer a subject's own immune cells to fight bloodborne cancer. Our guest for this episode, Raquel Munoz from the Hospital Universitario Virgen del Rocío in Seville Spain, is doing research in this exciting CAR-T cell therapy space. Specifically, she is working to develop methods to help better quantify and understand the expansion of CAR-T cells in the body to help monitor treatment and predict outcomes.  We learn about why digital PCR was selected for her work and how it's helped raise confidence in the results they're getting. We even hear about how she believes this treatment will find success in treating solid tumor cancers.In Cassie's career corner, we learn how Raquel found her career path and love of immunology and working in a hospital setting. Raquel also shares some great career advice, stories of lab mishaps, and the dangerous hobby that she says is some of the only time she's not thinking about work or problems. Visit the Absolute Gene-ius page to learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System. 

BUFFALO, NY- December 5, 2023 – A new #researchpaper was #published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 15, Issue 22, entitled, “Contribution of membrane raft redox signalling to visfatin-induced inflammasome activation and podocyte injury.” The number of obese patients with end stage renal disease has increased significantly worldwide in the last few decades. Obesity results in an increased risk for chronic kidney diseases like diabetes and hypertension which consequently result in chronic kidney disease or even end-stage renal disease. However, the exact mechanism of how obesity increases the advancement of chronic kidney disease is still uncertain. Recently, researchers Saisudha Koka, Sreenidhi Surineni, Gurinder Bir Singh, and Krishna M. Boini from the University of Houston, Texas A&M University and the University of California Riverside have shown that adipokine visfatin-induced NLRP3 inflammasome activation contributes to podocyte injury. However, the molecular mechanisms of how visfatin induces the Nlrp3 inflammasome activation and podocyte damage is still unknown. The present study tested whether the membrane raft (MR) redox signaling pathway plays a central role in visfatin-induced NLRP3 inflammasomes formation and activation in podocytes. “In this study, it is proposed that visfatin induces the NLRP3 inflammasome activation in podocytes, leading to glomerular inflammatory injury in the kidney and the development of CKD, may be primarily driven by NADPH oxidase-mediated membrane raft redox signalling.” Upon visfatin stimulation, an aggregation of NADPH oxidase subunits, gp91phox and p47phox, was observed in the MR clusters, forming an MR redox signaling platform in podocytes. The formation of this signaling platform was blocked by prior treatment with MR disruptor MCD or NADPH oxidase inhibitor DPI. In addition, visfatin stimulation significantly increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1, IL-β production, cell permeability in podocytes compared to control cells. Pretreatment with MCD, DPI, WEHD significantly abolished the visfatin-induced colocalization of NLRP3 with Asc or NLRP3 with caspase-1, IL-1β production and cell permeability in podocytes. Furthermore, Immunofluorescence analysis demonstrated that visfatin treatment significantly decreased the podocin and nephrin expression (podocyte damage) and prior treatments with DPI, WEHD, MCD attenuated this visfatin-induced podocin and nephrin reduction. In conclusion, their results suggest that visfatin stimulates membrane raft clustering in the membrane of podocytes to form redox signaling platforms by aggregation and activation of NADPH oxidase subunits enhancing O2·− production, leading to NLRP3 inflammasome activation in podocytes and ultimate podocyte injury. “Through experiments conducted on cultured podocytes, we have demonstrated, for the first time that membrane raft-associated redox signalling is essential for the NLRP3 inflammasomes assembly and activation in response to visfatin, subsequently resulting in podocyte dysfunction and injury. These findings shed light on a novel mechanism underlying inflammasome activation and injury of podocytes triggered by visfatin.” DOI - https://doi.org/10.18632/aging.205243 Corresponding author - Krishna M. Boini - kmboini@uh.edu Video short - https://www.youtube.com/watch?v=cIzaHj31GBo Visit our website at https://www.Aging-US.com​​ and connect with us: SoundCloud - https://soundcloud.com/Aging-Us Facebook - https://www.facebook.com/AgingUS/ X - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ MEDIA@IMPACTJOURNALS.COM

BUFFALO, NY- October 18, 2023 – A new editorial paper was published in Oncotarget's Volume 14 on April 14, 2023, entitled, “The nuclear envelope and metastasis.” In their new editorial, researchers Emily Hansen and James M. Holaska from Rowan University discuss nuclear morphology — one of the basic visual criteria used by pathologists to diagnose breast cancer. Immunofluorescence staining of the nuclear structural proteins lamin B and emerin was recommended as an effective diagnostic tool for both thyroid and breast cancer, suggesting nuclear structure is intimately tied to malignant transformation. But what role nuclear morphology plays in cancer transformation and progression remains unclear. “The most likely explanation for why cancer cells present with distinct nuclear morphology is thought to be related to the most likely route of cancer spread: the vasculature.” For a tumor to metastasize, cancer cells need to enter and exit the blood and lymphatic vessels by squeezing through extremely small gaps in the endothelium, most of which are 1.2–2 µm in diameter. While the cytoplasm is very flexible and the cytoskeleton can rearrange to fit through openings as narrow as 1 µm, the nuclear diameter (10–20 µm) and its considerable stiffness (2–10x stiffer than the cytoplasm) represent physical barriers to this process. “Thus, to enable metastasis, cancer cells must also increase their nuclear malleability.” Studies have shown that nuclear softening is associated with tumor aggressiveness and metastasis. Although nuclear softening is one of the ‘hallmarks of cancer' it remains poorly understood. Nuclear shape and stiffness are governed by a complex set of structural proteins that serve as both scaffolds and signaling proteins to influence almost all aspects of nuclear function. The best studied nucleostructural proteins are lamins, which are frequently downregulated in cancer. However, it is difficult to ascertain whether specific functional consequences are due to lamins or due to displacement of lamin-interacting proteins upon lamin loss. For example, nuclear size and shape is also governed by emerin, which binds to lamins at the nuclear envelope (NE) and upon lamin loss is retained in the endoplasmic reticulum. Like lamins, emerin is frequently mutated in cancer, with mutations in its transmembrane and actin-binding domains. “We found that in breast cancer, emerin expression in tumor tissue is significantly correlated to survival time [16]. These data suggest emerin plays a central role in pathogenic transformation and progression of malignant breast tissue.” DOI - https://doi.org/10.18632/oncotarget.28375 Correspondence to - James M. Holaska - holaska@rowan.edu Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28375 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, emerin, metastasis, mechanotransduction, breast cancer, nucleoskeleton About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.16.525994v1?rss=1 Authors: Luo, Y., Li, J., Xiong, Y. Abstract: Purpose: Glioblastoma (GBM) is the most aggressive and common form of brain cancer in adults. GBM is characterised by poor survival as the lack of effective therapies. This research aims to detect the roles of SNAREs in GBM and improve our knowledge of targeting therapy for GBM. Materials and methods: the expression of SNAREs and their correlation with overall survival (OS) in GBM are investigated using the GEPIA. The level of BET1 in GBM cell lines was tested by RT-qPCR, and its biological functions in GBM cells were tested by Transwell assay and CCK8 kit. The effect of BET1 on the location of MMP14 is identified by Immunofluorescence. Results: The expression profile of SNARE family members in GBM tissue is changed dramatically. Among them, the mRNA levels of BET1 and VAMP3 are up-regulated, and their expression negatively correlates with OS. BET1 is also increased in GBM Cell Lines, and it is required for efficient GBM cell migration and invasion partly because it mediates the transport of MMP14 to the plasma membrane. Conclusion: GBM has highly diffusive and infiltrative ability in nature, making complete surgical resection almost impossible. Our data shows that BET1 is highly expressed in GBM tissue, negatively correlated with OS, and essential for GBM cell migration and invasion. These results indicate that SNARE BET1 may present a potential target for GBM treatment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.09.523293v1?rss=1 Authors: Chiu, K., Berrada, Y., Eskndir, N., Song, D., Fong, C., Naughton, S., Chen, T., Moy, S., Gyurmey, S., James, L., Ezeiruaku, C., Capistran, C., Lowey, D., Diwanji, V., Peterson, S., Parakh, H., Burgess, A., Probert, C., Zhu, A., Anderson, B., Levi, N., Gerlitz, G., Packard, M. C., Dorfman, K. A., Bahiru, M. S., Stephens, A. D. Abstract: Mitosis is an essential process in which the duplicate genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.22.521561v1?rss=1 Authors: Niwa, S., Chiba, K. Abstract: Kinesin-1, a motor protein composed of the kinesin heavy chain (KHC) and the kinesin light chain (KLC), is fundamental to cellular morphogenesis and function. A monoclonal antibody (mAb) called H2 recognizes the KHC in a broad range of species and is one of the most widely used mAbs in cytoskeletal motor research. Here, we generated vectors that expressed recombinant H2 in mammalian cells. We demonstrated that the recombinant H2 performed as well as the hybridoma-derived H2 in western blotting and immunofluorescence assays. The recombinant H2 could detect all three human KHC isotypes (KIF5A, KIF5B, and KIF5C) and amyotrophic lateral sclerosis (ALS)-associated KIF5A aggregates in the cell. Immunofluorescence microscopy showed that the single chain variable fragment (scFv) derived from the H2 mAb could specifically recognize KHCs in cells. In addition, we developed a chickenized anti-KHC scFv(H2), which broadens the application of H2 in immunofluorescence microscopy. Collectively, our findings validate recombinant H2 as useful for studying the function of KHCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.22.517521v1?rss=1 Authors: Azbazdar, Y., Poyraz, Y. K., Ozalp, O., Nazli, D., Ipekgil, D., Cucun, G., Ozhan, G. Abstract: Non-alcoholic fatty liver disease (NAFLD) includes a range of liver conditions ranging from excess fat accumulation to liver failure. NAFLD is strongly associated with high-fat diet (HFD) consumption that constitutes a metabolic risk factor. While HFD has been elucidated concerning its several systemic effects, there is little information about its influence on the brain at the molecular level. Here, by using a high-fat diet (HFD)-feeding of adult zebrafish, we first reveal that excess fat uptake results in weight gain and fatty liver. Prolonged exposure to HFD induces a significant increase in the expression of pro-inflammation, apoptosis, and proliferation markers in the liver and brain tissues. Immunofluorescence analyses of the brain tissues disclose stimulation of apoptosis and widespread activation of glial cell response. Moreover, glial activation is accompanied by an initial decrease in the number of neurons and their subsequent replacement in the olfactory bulb and the telencephalon. Long-term consumption of HFD causes activation of Wnt/Beta-catenin signaling in the brain tissues. Finally, fish fed an HFD induces anxiety, and aggressiveness and increases locomotor activity. Thus, HFD feeding leads to a non-traumatic brain injury and stimulates a regenerative response. The activation mechanisms of a regeneration response in the brain can be exploited to fight obesity and recover from non-traumatic injuries. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Even though tissues are tridimensional structures, most tissue research is done on two-dimensional tissue slides. This leaves a tremendous amount of biological information on the table. This episodes' guest - Sharla White, Ph.D., the vice president of research and development at ClearLight Biotechnologies explains how tissue clearing and 3D immunofluorescence can take your tissue research to a whole new level. With the rise of immuno-oncology, the importance of immune cell interactions with the tumor cells is now routinely interrogated with immunofluorescent markers the spatial relationships of different immune cell populations are investigated. But how can we investigate something happening in a 3D space on a flat, two-dimensional tissue section? The truth is - in a very restricted manner. This is where tissue clearing and 3D immunofluorescence come into play. The tissue clearing technology -CLARITY, developed by ClearLight Biosciences allows for maintaining the integrity of tissue and visualizing cells in their original place and shape at the same time by using 3D immunofluorescence. In order to image deeper (beyond 100 micrometers), the light-scattering lipids of the tissue need to be removed and the refractive indexes of collagen, bone, and other tissue components need to be aligned. This is done after fixing the tissue and embedding it in a hydrogel. It ensures that the tissue structure is maintained before the detergent is applied to wash out the light-scattering lipids. Once tissue clearing is done, antibodies with properties and in amounts compatible with the process are used for 3D immunofluorescence.  This powerful technology does not come without challenges such as:the necessity of tissue bleaching for melanoma samples,selection of appropriate immunofluorescence markers,size of the 3D image files generated for visualization (often as big as 500 gigabytes reaching terabytes of data!)meaningful interpretation of the results  Listen to the full episode to learn how Dr. White's team is approaching all the challenges, leveraging CLARITY potential and how this technology changes the way we do tissue research.  This episode's resources:ClearLight YouTube channel ClearLight Biotechnologies website Original clarity paper "Structural and molecular interrogation of intact biological systems" Imaris -  software for 3D visualization of tissue specimen Episodes you might also like:Immuno-oncology 101 w/ Elfriede Noessner, HelmholzZentrum, Visiopharm AdvisorIntroduction to multiplex for tissue image analysis (Part 1) w/ Regan Baird, Visiopharm

New research on the Omicron variant unpicked by James Gallagher, BBC health and science correspondent. Plus many people listen to music for hours every day, and often near bedtime in the hope of a good night's sleep. But if you can't get the tune out of your head could this be counter-productive? In new research, neuropsychologist Michael Scullin of Baylor University has looked at the rarely studied effect of these so called earworms. And could fish oils one day be used to treat some forms of severe depression? Claudia hears from Alessandra Borsini of King's College London who has been examining the impact of omega-3 polyunsaturated fatty acids in the lab and has followed up with a promising trial on severely depressed patients. Plus James Gallagher explains that despite there being no evidence 5G mobile networks are harmful many types of necklaces and accessories claiming to "protect" people from 5G have hit the market. Now the Dutch authority for nuclear safety and radiation protection warns that with long term use such anti-5G products themselves could be harmful due to radioactive concerns. Presenter: Claudia Hammond Producer: Erika Wright (Picture: Omicron variant (B.1.1.529): Immunofluorescence staining of uninfected and infected Vero E6 cells. Photo credit: Microbiology HKU/BSIP/Universal Images Group via Getty Images.)

New research on the Omicron variant unpicked by James Gallagher, BBC health and science correspondent. Plus many people listen to music for hours every day, and often near bedtime in the hope of a good night’s sleep. But if you can’t get the tune out of your head could this be counter-productive? In new research, neuropsychologist Michael Scullin of Baylor University has looked at the rarely studied effect of these so called earworms. And could fish oils one day be used to treat some forms of severe depression? Claudia hears from Alessandra Borsini of King’s College London who has been examining the impact of omega-3 polyunsaturated fatty acids in the lab and has followed up with a promising trial on severely depressed patients. Plus James Gallagher explains that despite there being no evidence 5G mobile networks are harmful many types of necklaces and accessories claiming to "protect" people from 5G have hit the market. Now the Dutch authority for nuclear safety and radiation protection warns that with long term use such anti-5G products themselves could be harmful due to radioactive concerns. Presenter: Claudia Hammond Producer: Erika Wright (Picture: Omicron variant (B.1.1.529): Immunofluorescence staining of uninfected and infected Vero E6 cells. Photo credit: Microbiology HKU/BSIP/Universal Images Group via Getty Images.)

A study from Hong Kong university shows Omicron replicates 70 times faster than two earlier variants of the SARS-Cov-2 virus. Virologist Malik Peiris, explains how tests using cells from the wind pipe showed the dramatic difference, which supports observations of increased transmission. In contrast Omicron replicated less well than other variants on cells from dep in thre lung – offering some possibility that it may produce mild infections. Tornados in the US do not normally occur in December. The one which swept across Kentucky and 3 other states was fuelled by weather patterns likely to have been influenced by long term climate change says Geographer James Elsner of Florida State University. The Parker Solar probe continues its mission of flying closer and closer to the sun. Results just published show what the data the probe picked up when it dipped into the surrounding plasma. NASA's Nicky Fox is our guide. And how many legs does a millipede have? Until now not as many as you might think. Entomologist Paul Marek of Virginia Tech reveals the Australian specimen with more legs than ever seen before. As many of us gear up for the annual Christmas feast, some of you may be wondering how to eat everything before it goes off. It's a great question, as the UN puts global food waste at a whopping 1.3 billion tonnes a year – that's one third of all edible produce being thrown in the bin. So this week the team investigates listener Peter's query about what makes some fruit and vegetables rot faster than others. Preserving food used to be about ensuring nomadic populations could keep moving without going hungry, but these days some things seem to have an almost indefinite shelf-life. Is it about better packaging or can clever chemistry help products stay better for longer? A Master Food Preserver explains how heat and cold help keep microbes at bay, and how fermentation encourages the growth of healthy bacteria which crowd out the ones that make us ill. Presenter Datshiane Navanayagam learns how to make a sauerkraut that could keep for weeks, and investigates the gases that food giants use to keep fruit and veg field-fresh. But as the industry searches for new techniques to stretch shelf-life even further could preservatives in food be affecting our microbiome? Research shows sulphites may be killing off ‘friendly' gut bacteria linked to preventing conditions including cancer and Crohn's disease. (Image: Omicron variant (B.1.1.529): Immunofluorescence staining of uninfected and infected Vero E6 cells. Credit: Microbiology HKU/BSIP/Universal Images Group via Getty Images)

A study from Hong Kong university shows Omicron replicates 70 times faster than two earlier variants of the SARS-Cov-2 virus. Virologist Malik Peiris, explains how tests using cells from the wind pipe showed the dramatic difference, which supports observations of increased transmission. In contrast Omicron replicated less well than other variants on cells from dep in thre lung – offering some possibility that it may produce mild infections. Tornados in the US do not normally occur in December. The one which swept across Kentucky and 3 other states was fuelled by weather patterns likely to have been influenced by long term climate change says Geographer James Elsner of Florida State University. The Parker Solar probe continues its mission of flying closer and closer to the sun. Results just published show what the data the probe picked up when it dipped into the surrounding plasma. NASA's Nicky Fox is our guide. And how many legs does a millipede have? Until now not as many as you might think. Entomologist Paul Marek of Virginia Tech reveals the Australian specimen with more legs than ever seen before. (Image: Omicron variant (B.1.1.529): Immunofluorescence staining of uninfected and infected Vero E6 cells. Credit: Microbiology HKU/BSIP/Universal Images Group via Getty Images) Presenter: Roland Pease Producer: Julian Siddle

Bienvenue sur RARE à l'écoute, la chaîne de Podcast dédiée aux maladies rares. Pour le quatrième épisode sur les amyloses cardiaques, nous recevons le Dr Fabien Le Bras, hématologue, praticien hospitalier au sein de l'unité hémopathie lymphoïde de l'hôpital Henri-Mondor à Créteil, qui travaille en coordination avec les cardiologues du Centre de Référence National des Amyloses Cardiaques. Nous abordons aujourd'hui la physiopathologie de l'amylose AL, le diagnostic et le typage de cette amylose, la prise en charge de l'amylose AL et des rechutes éventuelles et l'importance de la coordination entre hématologues et cardiologues. Si vous désirez vous informer et aller plus loin dans la connaissance de cette pathologie, nous vous donnons rendez-vous sur notre site internet www.rarealecoute.com. L'orateur n'a reçu aucune rémunération pour la réalisation de cet épisode.   Invité : Dr Fabien Le Bras – Hôpital Henri-Mondor - Créteil http://www.reseau-amylose-chu-mondor.org/index.php/78-reseau-amylose-chu-henri-mondor/24-site-internet-du-centre-de-reference-des-amyloses-cardiaques  L'équipe : Virginie Druenne - Programmation Cyril Cassard - Animation Hervé Guillot - Production Crédits : Sonacom

This episode is brought to you by Visiopharm.In this third and last episode of the multiplex mini-series with Regan Baird from Visiopharm we look at the considerations when choosing an image analysis software for phenotyping.The two main points to consider when choosing phenotyping image analysis software are segmentation assistance and data visualization. Segmentation assistance:Before different markers are attributed to different cells in the tissue and cell phenotypes are determined, cell boundaries need to be delineated. The automatic delineation of these boundaries by image analysis software is called cell segmentation. Cells in tissue slides can have different shapes and sizes, which depend on the plane of sectioning, heterogeneity of the investigated tissue, and the disease stage. This makes the task of segmentation challenging. Unlike in single-cell confocal microscopy images, where the cell borders are very well-demarcated, in tissue they often need to be estimated. A separate segmentation (e.g., membrane) marker can help significantly, but a perfect cell segmentation is not attainable.To best estimate the cell boundaries, rule-based classical computer vision approaches or artificial intelligence (AI) – powered approaches can be used. In rule-based approaches, we are working with well-defined features on which the segmentation is based, but we need to make concessions. The AI-powered models are only as good as the examples we train the models on. To combine the advantages of both, Visiopharm offers an AI-based nuclear segmentation as the starting point and a rule-based and marker-based second step to obtain the most reliable cell segmentation for phenotyping. Data visualization:The adequate visualization and handling of the obtained data depend on the software used. To understand and interpret the multidimensional multiplex and phenotyping data we need to interpret graphs, plots, two-dimensional reduction plots, and other data visualizations for all the images in multiplex studies. In order to evaluate how well the phenotyping has performed and to export meaningful results, the correct visualization tools need to be used.If you need assistance or have questions about multiplexing and phenotyping visit the Visiopharm’s website and contact the Visiopharm team. This episode’s resources:Multiplexing mini-series Part 1: Introduction to multiplex for tissue image analysis (part 1) w/ Regan Baird, VisiopharmMultiplexing mini-series Part 2: How to make sense of multiplex data with phenotyping? (part 2) w/ Regan Baird

This episode is brought to you by Visiopharm.Multiplex tissue staining can generate large amounts of data to help identify distinct information about particular cells in tissue. Immuno-oncology is a field where it is common practice to use multiplexing, in particular for cell phenotyping in tissue.Phenotyping is the ability to classify every individual cell in the tissue based on the biomarker panel used. The panels are designed to identify cells of different lineages as well as cell activation states within each lineage, which is of utmost importance for the personalized therapeutic approaches in oncology. Although multiplex data can be visualized manually, e.g., by switching on and off different fluorescence channels, its interpretation requires computational assistance. If the multiplex assay only contains a few markers, the rules for detecting potential phenotypes can be designed manually, but as the number of markers increases the number of potential phenotypes increases exponentially. In order to sort through the cellular phenotypes in higher-plexes, machine learning-based auto clustering has been implemented. This method is based on the way cells are characterized in flow cytometry and has been adapted to automatically identify phenotypes of cells in tissue images. The adequate visualization and handling of the generated data depend on the software used. In the next episode, we will be talking about the considerations when choosing an image analysis software program for phenotyping. To learn more visit Visiopharm’s websiteThis episode’s resources:Multiplexing mini-series Part 1: Introduction to multiplex for tissue image analysis (part 1) w/ Regan Baird, Visiopharm

This episode is brought to you by Visiopharm.After experimenting with multidimensional, multimarker, and multicolor single-cell imaging modalities during his postdoc at Beth Israel Deaconess Medical Center in Boston, looking at 2D images of tissue stained just with hematoxylin and eosin (H&E) seemed to him a bit simplistic…and then he was tasked with doing tissue image analysis (IA). When relying just on H&E, IA can be a very challenging task. So, to both simplify it and extract more information from the tissue, multiplex staining can be implemented.  In this three-part episode miniseries Regan Baird, Ph.D., scientific sales manager at Visiopharm introduces us to the concepts of multiplexing and cell phenotyping as well as to image analysis approaches relevant for multiplex data analysis.Multiplexing in the context of life sciences is referred to as taking multiple measurements at the same time on the same specimen. With tissue slides the easiest method of multiplexing is immunohistochemistry (IHC) based virtual multiplexing where consecutive sections of tissue are stained with a single IHC marker and later each slide is imaged and co-registered to simulate the presence of several IHC markers in the tissue of interest. More complicated, but more precise methods allowing for visualizing cellular colocalization of biomarkers include multicolor bright field IHC (visualizing up to five biomarkers per tissue but colocalizing reliably a maximum of only two biomarkers per cell), immunofluorescence (IF) potentially with spectral unmixing, to increase the number of biomarkers per tissue section as well as per cell to nine, and imaging mass cytometry where instead of chromogens or fluorophores heavy metals are used, which increases the number of biomarkers up to 60 in a single section of tissue.All these multiplex modalities have their advantages and disadvantages, and the choice of the appropriate method should be guided by the design of the experiment as well as scientific and/ or diagnostic questions we want to address.For example, currently a widely used application of IF multiplexing is phenotyping cells in the tissue. This not only allows for the characterization of single cells but also lets us interrogate and investigate spatial relationships between different cell populations giving us information about the interactome of different cells and the environment in which they live. To learn more about phenotyping join us for the next episode of the Multiplexing Miniseries next week.This episode’s resources:Multiplexing mini-series Part 2: How to make sense of multiplex data with phenotyping? (part 2) w/ Regan BairdVisiopharmTop 20 Pathology podcasts you must follow in 2021 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384354v1?rss=1 Authors: Geng, J., Liu, G., Wu, Y., Kong, F., Ma, S., Fu, L. Abstract: Multiple sclerosis (MS) is a complex, progressive neuroinflammatory disease associated with autoimmunity. Currently, effective therapeutic strategy was poorly found in MS. Experimental autoimmune encephalomyelitis (EAE) is widely used to study the pathogenesis of MS. Previous studies have shown that bone marrow mesenchymal stem Cells (BMSCs) transplantation could treat EAE animal models, but the mechanism was divergent. Here, we systematically evaluated whether BMSCs can differentiate into neurons, astrocytes and oligodendrocytes to alleviate the symptoms of EAE mice. We used Immunofluorescence staining to detect MAP-2 neurons marker, GFAP astrocytes marker, and MBP oligodendrocytes marker expression to evaluate whether BMSCs can differentiate. The effect of BMSCs transplantation on inflammatory cell invasion and demyelination in EAE mice were detected by Hematoxylin-Eosin (H&E) and Luxol Fast Blue (LFB) staining. Inflammatory factors expression was detected by ELISA and RT-qPCR. Our results showed that BMSCs could be induced to differentiate into neuron cells, astrocytes and oligodendrocyte in vivo and in vitro. In addition, BMSCs transplant improved the survival rate and weight, and reduced neurological function scores and disease incidence of EAE mice. Moreover, BMSCs transplant alleviated the inflammation and demyelination of EAE mice. Finally, we found that BMSCs transplantation down-regulated the expression levels of pro-inflammatory factors TNF-, IL-1{beta} and IFN-{gamma}, and up-regulated the expression levels of anti-inflammatory factors IL-10 and TGF-{beta}. In conclusion, this study found that BMSCs could alleviate the inflammatory response and demyelination in EAE mice, which may be achieved by the differentiation of BMSCs into neurons, astrocytes and oligodendrocytes in EAE mice. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.25.334201v1?rss=1 Authors: Chang, C.-P., Chang, Y.-G., Chuang, P.-Y., Nguyen, A. T. N., Chou, F.-Y., Cheng, S.-J., Chen, H.-M., Jin, L.-W., Carvalho, K., Huin, V., Buee, L., Blum, D., Liao, Y.-F., Lin, C.-J., Chern, Y. Abstract: Tau hyperphosphorylation favors the formation of neurofibrillary tangles and triggers the gradual loss of neuronal functions in tauopathies, including Alzheimer's disease. Herein, we demonstrated that chronic treatment with an inhibitor (J4) of equilibrative nucleoside transporter 1 (ENT1), which plays a critical role in controlling adenosine homeostasis and purine metabolism in the brain, exerted beneficial effects in a mouse model of tauopathy (Thy-Tau22, Tau22). Chronic treatment with J4 improved spatial memory deficits, mitochondrial dysfunction, synaptic plasticity impairment, and gliosis. Immunofluorescence assays showed that J4 not only reduced Tau hyperphosphorylation but also normalized the reduction in mitochondrial mass and suppressed the abnormal activation of AMP-activated protein kinase (AMPK), a pathogenic feature that is also observed in the brains of patients with tauopathies. Given that AMPK is an important energy sensor, our findings suggest that energy dysfunction is associated with tauopathy and that J4 may exert its protective effect by improving energy homeostasis. Bulk RNA-seq analysis revealed that J4 also mitigated immune signature associated with Tau pathology including C1q upregulation and A1 astrocyte markers. Collectively, our findings suggest that identifying strategies for normalizing energy and neuroimmune dysfunctions in tauopathies through adenosinergic signaling modulation may pave the way for the development of treatments for Alzheimer's disease. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318683v1?rss=1 Authors: Sanchez-Lanzas, R., Castano, J. G. Abstract: DJ-1/PARK7 mutations are linked with familial forms of early onset Parkinson disease (PD). We have studied the degradation of untagged DJ-1 WT and missense mutants in mouse embryomic fibroblasts obtained from DJ-1 null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants: L10P, M26I, A107P, P158DEL, L166P, E163K and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E and A179T are as stable as the DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158DEL, L166P, E163K and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of those mitochondrialy associated DJ-1 mutants, but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.289256v1?rss=1 Authors: Knabbe, J., Protzmann, J., Schneider, N., Dannehl, D., Berger, M., Wei, S., Strahle, C., Jaiswal, A., Lugani, S., Zheng, H., Krueger, M., Rohr, K., Spanagel, R., Scholz, H., Bilbao, A., Engelhardt, M., Cambridge, S. Abstract: Alcohol intoxication at early ages is a risk factor for development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope labeled mice combined with quantitative mass spectrometry to screen over 2000 hippocampal proteins of which 72 changed synaptic abundance up to two-fold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and Ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naive mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila. This introduces mitochondrial trafficking as a process implicated in reward learning, and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior. Copy rights belong to original authors. Visit the link for more info

kyonさんをゲストに迎え、自作のモノクローナル抗体をつくるプロトコルとその魅力について教えてもらいました。Shownotes 抗体 (antibody)…免疫グロブリンの総称。参考ページ:抗体とは (MBLのwebページ)、抗体とは？ (中外製薬webページ) 抗体ガイド 4. ポリクローナル抗体とモノクローナル抗体 (abcamのwebページ) モノクローナル抗体の作り方 (中外製薬webページ) 重鎖・短鎖…抗体を構成する部位。参考ページ: Sigmaのwebページ エピトープ…抗体の抗原認識部位 免疫染色…抗体を用いて目的のタンパク質のみを染めることで可視化する手法。最も一般的なのは蛍光でラベルする蛍光免疫染色 (Immunofluorescence staining) ELISA ウェスタンブロッティング 免疫沈降 (Immunoprecipitation) ミエローマ…マウス骨髄腫由来の細胞株。免疫グロブリン (抗体) を大量に生産する B細胞…免疫機構を担うリンパ球の一種 細胞融合…細胞同士の膜の融合によって、2つの細胞を１つにする技術。細胞は核も融合し染色体数が4倍体になるが、徐々に減少し、あるレベルで安定化し増殖する細胞株となる (実験医学webページより一部改変して抜粋) ハイブリドーマ…B細胞とミエローマが融合した細胞 PIWI family…kyonが研究対象にしている遺伝子。詳細はep.14へGO! アジュバント…ワクチンと一緒に投与することで、免疫効果を高める Hamers-Casterman et al., Nature (1993) … ラクダ科の動物の抗体について初めて報告した論文 蛇毒抗体の人 ポリエチレングリコール (PEG) (Wikipedia) エレクトロポレーション…電気穿孔法ともいう。電流パルスにより細胞に物理的な刺激を与え、細胞膜を一時的に破壊し透過性を上げることで、目的の物質を能動的に入れ込む技術 Neon™ Transfection System … 針みたいなエレポ MIHS法…横浜国立大学の栗原靖之先生のグループが開発したハイブリドーマの作製方法 Editorial notes 完全に余談ですが、実験のやる気がでない日はこの動画が脳内再生されます。Forget about doing a western blot~♪ (kyon) いつか抗体も自作したくなった (Soh) バンバン抗体つくりましょう。途中だいぶぼんやりしていて的外れな質問をしてしまった…(細胞融合もうちょい話したかった感)(tadasu) 抗体と言えばブレンパワードのアンチボディ格好良いですよね(coela)

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.16.206094v1?rss=1 Authors: Goksu Erol, A. Y., Akinci, E., Kocanci, F. G., Akcakale, F., Demir Dora, D., Uysal, H. Abstract: Introduction: Microglia secretome includes not only growth factors and cytokines which support neuronal survival, it includes neurotoxic cytokines/enzymes, as well. MPTP is a neurotoxin which has degenerative effects on SH-SY5Y neuroblastoma cells. Masitinib mesylate is a tyrosine kinase inhibitor which has been shown to have beneficial effects in neurodegenerative diseases. Aim : We first aimed to determine the most efficient microglial cell conditioned medium in terms of neurodegenerative effect. Next, we investigated the possible protective/therapeutic effects of masitinib against MPTP/microglia-induced degeneration of differentiated ( d )-SH-SY5Y cells, and the role of transforming growth factor (TGF)-b1 and nitric oxide (NO) in these events. Material-Methods : Non-stimulated/LPS-stimulated microglia cells were treated with masitinib or its solvent, DMSO. With or without MPTP- d -SH-SY5Y cell cultures were exposed to the conditioned media (CM) from microglia cell cultures, followed by cell survival analysis. Immunofluorescence staining of microglia and d -SH-SY5Y cells were performed with anti-CD-11b and anti-PGP9.5 antibody, respectively. TGF-b1/NO concentrations in CM of microglia/ d -SH-SY5Y cell culture were measured. Results: The initial 24 hrs CM of non-stimulated microglia cell culture was found to be the most detrimental microglial medium with lowest survival rates of treated d -SH-SY5Y cells. The toxicity of 48 and 72 hrs CM on d -SH-SY5Y cells were both lower than that of 24 hrs CM. Masitinib (0.5 {micro}M), significantly prevented MPTP-related cell degeneration of d -SH-SY5Y cells. It also decreased the degenerative effects of both non-induced/LPS-induced microglia CM on with or without MPTP- d -SH-SY5Y cells. Although NO levels in microglia CM showed a negative correlation with survival rates of treated d -SH-SY5Y cells, a positive correlation was seen between TGF-{beta}1 concentrations in microglial CM and rates of treated d -SH-SY5Y cell survival. Conclusion : Masitinib ameliorates viability of with/without MPTP- d -SH-SY5Y cells. It does not only reverse the degenerative effects of its solvent, DMSO, but also prevents the degenerative effects of microglial secretions and MPTP. We suggest that masitinib begins to act as a neuroprotective agent via mediating TGF-b1 and NO secretion, as neurons are exposed to over-activated microglia or neurotoxins. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.12.149062v1?rss=1 Authors: da Rocha, J. F., Bastos, L., Domingues, S. C., Bento, A. R., Konietzko, U., da Cruz e Silva, O. A. B., Vieira, S. I. Abstract: The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders like Schizophrenia and AD. Here we show that APP interacts with these two EGFR ligands and characterize the effects of APP-EGF interaction in ERK activation and neuritogenesis. HB-EGF was identified as a novel APP interactor in a yeast two-hybrid screen of a human brain cDNA library. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were also observed to synergistically activate the ERK signaling pathway, crucial for early neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in conditions of APP overexpression and EGF exposure, confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes per cell. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.14.095307v1?rss=1 Authors: Mirzaei, M., Gupta, V., Chitranshi, N., Deng, L., Pushpitha, K., Abbasi, M., Chick, J., Rajput, R., Wu, Y., McKay, M. J., Salekdeh, G. H., Gupta, V., Haynes, P. A., Graham, S. Abstract: Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 vs 20.8 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using multiplexed quantitative proteomics approach. Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (less than equal to 0.83) while expression of 109 proteins was upregulated (more than equal to 1.2-fold change) under glaucoma conditions (less than equal to 0.05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton and actin filament organisation, along with increased expression coagulation cascade, apoptosis, oxidative stress and RNA processing markers. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins, were exclusive to the human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP. Copy rights belong to original authors. Visit the link for more info

The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. It has evolved a range of mechanisms to aid in colonization and persistence, manipulating the host immune response to avoid clearance. A major factor in this is the secreted vacuolating cytotoxin VacA which has a variety of effects on host cells. VacA is endocytosed and forms anion-selective channels in the endosome membrane, causing the compartment to swell. The resulting VacA-containing vacuoles (VCVs) can take up most of the cellular cytoplasm. Even though vacuolation is VacA's most prominent and namesake effect, the purpose of the vacuoles is still unknown. VacA exerts influence on the host immune response in various ways, both pro- and anti- inflammatorily. Most importantly, it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell activation and proliferation and thereby suppressing the host immune response. Furthermore, VacA is transported to mitochondria, where it activates the mitochondrial apoptosis pathway. Within the cell, VacA has only been shown to localize to endocytic compartments/VCVs and mitochondria. Considering its diverse effects, however, the existence of other cellular sites of action seems plausible. In this study, the VCV proteome was comprehensively analyzed for the first time in order to investigate VCV function. To this end, three different strategies for VCV purification from T-cells were devised and tested. Eventually, VCVs were successfully isolated via immunomagnetic separation, using a VacA-specific primary antibody and a secondary antibody coupled to magnetic beads. The purified vacuoles were then measured by mass spectrometry, revealing not only proteins of the endocytic system, but also proteins usually localized in other cellular compartments. This apparent recruitment of proteins involved in all kinds of cellular pathways indicates a central function of VCVs in VacA intoxication effects. In a global evaluation, the VCV proteome exhibited an enrichment of proteins implicated in immune response, cell death, and cellular signaling; all of these are processes that VacA is known to influence. One of the individual proteins contained in the sample was STIM1, a calcium sensor normally residing in the endoplasmic reticulum (ER) that is important in store- operated calcium entry (SOCE). This corroborates the findings of a concurrent report, in which VacA severely influenced SOCE and colocalized with STIM1. A direct interaction of STIM1 with VacA was examined in a pull-down assay, but could be neither shown nor excluded. Immunofluorescence experiments conducted in HeLa cells confirmed the presence of VacA in the ER and also found it to traffic to the Golgi apparatus, identifying these two cellular compartments as novel VacA target structures. The exact route of VacA transport remains unclear, but the involvement of both the ER and the Golgi suggests the possibility of retrograde trafficking, analogous to other bacterial toxins like shiga and cholera toxins. In summary, the elucidation of the VCV proteome and the discovery of the ER and the Golgi apparatus as VacA target structures have generated intriguing starting points for future studies. The detection of many proteins implicated in VacA intoxication effects in the VCV proteome leads to the proposal of VCVs as signaling hubs that may coordinate the complex meshwork of VacA effects. Further investigation of individual proteins is expected to help greatly in illuminating this matter.

Actin is one of the most abundant proteins in eukaryotic cells and regulation of the microfilament system is crucial for a wide range of cellular functions including cell shape, cell motility, cell division and membrane dynamics. The aim of this thesis was (1) to gain a better understanding of the function of distinct actin binding domains in the regulation of the actin cytoskeleton and (2) to elucidate the role of actin variants. WH2 domains (WH2, Wiskott-Aldrich syndrome protein homology 2) are ubiquitous multifunctional regulators of actin dynamics. The protein Spire contains four central WH2 domains A-B-C-D with about 20 amino acids each and the cyclase-associated protein CAP2 contains only one WH2 domain. Under certain conditions, they can (1) nucleate actin polymerization, (2) disintegrate actin filaments and (3) sequester actin monomers. Here, the influence of selected Drosophila melanogaster Spire-WH2 and Mus musculus CAP2-WH2 domain constructs on actin dynamics was tested in vitro. To act as a filament nucleator, at least two WH2 domains are required, and nucleation of actin polymerization was only observed at substoichiometric concentrations of WH2 domains over actin. At higher concentrations, the sequestering activity of WH2 domains takes over. Preformed and purified SpireWH2-actin complexes act as extremely efficient nuclei for actin polymerization, even at superstoichiometric WH2 concentrations, under which free WH2 domains would sequester actin. All analyzed constructs, including these with only a single WH2 domain, sequester actin as well as they can disrupt filaments. This latter and most peculiar behavior of WH2 domains was observed in fluorometric, viscometric and TIRF assays. The WH2 domains seem to have such a high affinity for actin that they can forcefully sequester monomers even from filaments and filament bundles, thus breaking the whole structures. Taken together, the data clearly show that SpireWH2-actin complexes are the intermediates that account for the observed nucleating activity, whereas free WH2 domains can disrupt filaments and filament bundles within seconds, again underlining the intrinsic versatility of this regulator of actin dynamics. These data have been confirmed by crystallography in collaboration with the groups of Prof. Dr. Tad Holak and Prof. Dr. Robert Huber (Martinsried, Germany). Besides the well-studied conventional actins many organisms harbor actin variants with unknown function. The model organism Dictyostelium discoideum comprises an actinome of a total of 41 actins, actin isoforms and actin-related proteins. Among them is filactin, a highly conserved actin with an elongated N-terminus. The 105 kDa protein has a distinct domain organization and homologs of this protein are present in other Dictyosteliidae and in some pathogenic Entamoebae. Here, the functions of filactin were studied in vivo and in vitro. Immunofluorescence studies in D. discoideum localize endogenous and GFP-filactin in the cytoplasm at vesicle-like structures and in cortical regions of the cell. A most peculiar behavior is the stress-induced appearance of full length filactin in nuclear actin rods. To perform in vitro analyses recombinant filactin was expressed in Sf9 cells. Fluorescence studies with the filactin actin domain suggest that it interferes with actin polymerization by sequestering G-actin or even capping filaments. Gel filtration assays propose a tetrameric structure of full length filactin. Protein interaction studies suggest that filactin is involved in the ESCRT (endosomal sorting complexes required for transport) pathway which is responsible for multivesicular body formation. The data on filactin suggest that only the conventional actins are the backbone for the microfilamentous system whereas less related actin isoforms have highly specific and perhaps cytoskeleton-independent subcellular functions.

The oculomotor system is one of the best studied motor systems. Afferents from a variety of premotor areas converge on the motoneurons in the three oculomotor nuclei to produce the different types of eye movements. All oculomotor motoneurons participate in all types of eye movements, and it was generally accepted, that these motoneurons form a relative homogenous group which provides the final common pathway for extraocular muscle (EOM)-motor innervation. The EOM in mammals, the effector organs of the oculomotor system, are fundamentally different from skeletal muscle. They have two functionally different layers, global and orbital layer, and are composed of two major muscle fibre classes, singly-innervated (SIF) and multiply innervated fibres (MIF). Previous studies in monkey revealed that SIF and MIF motoneurons are anatomically separated and have different premotor inputs, which support the idea of a dual motor innervation of EOM rather than a final common pathway from motoneuron to EOM. Up to date, neither motoneuron type has been further characterized nor has any study proven their presence in other species to support the hypothesis of the dual motor innervation as a common concept in mammals. The functional implication of this system remains speculative, though a role of MIFs together with their motoneurons in a sensory feedback system controlling the EOMs is quite possible and heavily debated. However, the lack of a common proprioceptor in eye muscles does not support this theory. In monkeys SIF and MIF motoneurons of extraocular muscles were identified by tracer injections into the belly or the distal myotendinous junction of the medial or lateral rectus muscle and further characterized by combined tracer detection and immunohistochemical methods. The experiments revealed that the MIF motoneurons in the periphery of the motor nuclei lack non-phosphorylated neurofilaments, parvalbumin and perineuronal nets, whereas SIF motoneurons intensively express all three markers. In addition to the histochemical differences, the MIF motoneurons are on average significantly smaller in size than the SIF motoneurons. Analogous to the study in monkey, the SIF and MIF motoneurons of the medial and lateral rectus muscle of rats were identified with tracer injections and further characterized by immunolabelling. For the first time it was shown that both motoneurons types are present in rat as well. The MIF motoneurons lie mainly separated from the SIF motoneurons, and are different in size and histochemical properties. As in monkey, the smaller MIF motoneurons lack non-phosphorylated neurofilaments and perineuronal nets, both of which are definite markers for the larger SIF motoneurons. A possible proprioceptive control of eye movements requires the presence of proprioceptive structures. The palisade endings represent the best candidate for an EOM-proprioceptor. They were analysed using antibody stains against the synaptosomal associated protein of 25kDA, SNAP-25. With this robust method palisade ending-like structures were identified for the first time in the extraocular muscles of the rat. Furthermore the rat palisade endings show characteristics of sensory structures thereby supporting their role in proprioception. In conclusion, the EOM of both monkey and rat are innervated by two sets of motoneurons which differ in localization, morphology and molecular components. These findings further support the presence of a dual motor control of EOM that may apply widely to mammals, since it was verified in monkey and rat. Palisade endings are a ubiquitous feature of mammal EOM and most likely provide sensory information used for the proprioceptive control of eye movements.

Initiation of transcription by eukaryotic RNA polymerase II is finely controlled by a multitude of regulatory factors. Among them, the negative cofactor 2 (NC2), composed of the subunits NC2alpha and NC2beta, is able to bind directly to TBP-DNA complexes, preventing the assembly of the general transcription factors TFIIA and TFIIB. Despite extensive research on the negative and positive function of NC2, several questions concerning its regulation remain unexplored. In particular, localization and post-translational modifications are poorly understood. This work is the first to give some insights on the regulation of this factor. We present evidence that both subunits contain a nuclear localization signal (NLS) responsible for the accumulation of proteins in the nucleus. Immunofluorescence studies showed that NC2 dimer localizes exclusively in the nucleoplasm. However, the two subunits reveal characteristic and unique distribution patterns: NC2alpha is also found in the nucleoli, and NC2beta in small concentrations also in the cytoplasm. Moreover, we show that the two subunits already dimerize in the cytoplasm and are transported into the nucleus as a complex. Interestingly, both NLS are essential for import of the dimer. We also report for the first time several isoforms of both subunits. In vivo labeling experiments showed that NC2alpha is specifically hyperphosphorylated during mitosis. This modification does not impair its ability to dimerize with the partner and bind to TBP-DNA complexes, nor affects the stability of the complex. Furthermore, the phosphorylated protein maintains the ability to mobilize TBP on the DNA. These results suggest that NC2 is still bound to DNA during mitosis, in line with the idea that this factor keeps TBP stably associated to DNA.

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPSc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies (TSEs), which affect both, humans and animals. Human prion diseases occur in infectious, sporadic or genetic forms. The "protein only" hypothesis argues that the key event in the pathogenesis represents the conversion of the normal host protein, PrPc, into its pathogenic isoform PrPSc. Prion diseases have been associated with the accumulation of this abnormally folded protein and its neurotoxic effects. However, it is not known if PrPc loss of function is an important factor since the normal biological function of PrPc, a cell surface-anchored glycoprotein predominantly expressed in neuronal cells, and the cellular processes in which this protein is involved remain obscure. Recently, the human 37 kDa laminin receptor precursor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), was identified as a binding partner for the cellular prion protein in a yeast two-hybrid screen. In order to characterize the possible role of LRP/LR as a cell surface receptor for PrPc, cell culture studies were performed to investigate the cellular localization of PrP and LRP/LR and to analyse the binding and internalization behaviour of PrP depending on the presence of LRP/LR on the cell surface of neuronal and non-neuronal cells. Immunofluorescence analysis of non-permeabilized murine neuroblastoma cells demonstrated that PrP and LRP/LR co-localize on the surface of these cells. In addition, baby hamster kidney (BHK) cells transfected with recombinant Semlik-Forest virus RNAs overexpressed human PrP and human LRP at their cell surface, the latter one orientated as a type II transmembrane protein with its C-terminus outside and its N-terminus inside the cell. Co-localization of both proteins was observed on BHK cells co-transfected with LRP and PrP encoding recombinant SFV RNAs. Cell binding and internalization assays with recombinant human PrP demonstrated the LRP/LR-dependent binding and endocytosis of externally added human PrP. An increased, dose-dependent cell binding of recombinant PrP was demonstrated by BHK cells overexpressing full-length human LRP on their cell surface. Trypsin treatment of the cell surface revealed the LRP dependent internalization of GST-tagged and untagged, glycosylated PrP. In contrast to wild-type LRP, the expression of an LRP mutant lacking its transmembrane domain led to the secretion of this mutant from transfected BHK cells and totally abolished the binding and internalization of exogenous, recombinant PrP. This LRP mutant could function as a decoy recetor in therapy of TSEs. The strict LRP/LR specificity of the PrP binding to neuronal cells was verified by testing the displacement capacity of a series of different antibodies in the LRP-PrP binding reaction. Only LRP and PrP specific antibodies were able to block totally the binding of human GST-fused PrP to N2a and NT2 cells whereas various control antibodies used for competition showed no effect. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of cellular PrP to the 37-kDa/67-kDa LRP/LR. The relationship between the 37-kDa LRP and the 67-kDa high-affinity LR is unknown so far. Both forms were observed in plasma membrane fractions of N2a cells. We conclude from these data that the 37-kDa/67-kDa laminin receptor acts as the main cell surface receptor for PrP. High-level expression and purification of recombinant, glycosylated prion proteins in mammalian cells is essential for a better understanding of the physiological function of PrPc and biochemical processes responsible for prion diseases. Due to the presence of important organelles, membranes and other cellular cofactors which are necessary for the correct processing, trafficking and localization of prion proteins mammalian cell culture systems such as the Semliki-Forest virus (SFV) system allow the synthesis and characterization of wild-type as well as mutant PrP to get a better insight into the biology of these proteins. Therefore, the SFV system was used to generate recombinant highly glycosylated human wild-type and human disease-associated mutant prion proteins as well as FLAG-tagged human and bovine PrP in cultured BHK cells. Both mutated variants, which are related to the human prion diseases fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) reveal proteinase K (PK) resistance, one of the most typical biochemical properties characteristic for the infectious scrapie isoform of the prion protein. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP without any significant differences regarding the cellular distribution and expression level. In addition, FLAG-tagged prion proteins were expressed with high efficiency in BHK cells showing the typical glycosylation pattern allowing the rapid and simple purification via anti-FLAG antibody chromatography. PrP dimers could play an essential role in the PrPc to PrPSc conversion process and might be involved in PrP interspecies transmission. Recently, crystallization of the prion protein in a dimeric form was reported. Size exclusion chromatography showed that native soluble homogeneous FLAG tagged prion proteins from hamster, man and cattle expressed in the baculovirus system were predominantly dimeric. The PrP/PrP interaction was confirmed in rec. SFV-RNA transfected BHK cells co-expressing FLAG and oligohistidine tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. The identification of the 37-kDa/67-kDa laminin receptor as the receptor for the cellular prion protein might represent an important step for a better understanding of the molecular biology of prion diseases and might lead to the development of powerful therapeutics such as LRP/LR specific antibodies for the treatment of these unconventional diseases.

The etiology of Riedel's invasive fibrous thyroiditis (IFT) has remained obscure. This rare disorder has been confused in the past with the more common fibrous variant of Hashimoto's disease. The typical histological features of IFT, in particular the presence of an invasive fibrosclerotic process in conjunction with a prominent chronic inflammatory infiltrate, suggest that the release of fibrogenic cytokines and other factors from these cellular infiltrates may play an important role in the pathogenesis of this condition. Our observations in routinely processed tissue sections obtained from patients with documented IFT of striking tissue eosinophilia led us to hypothesize that eosinophils and their products may play a role in the evolution of this disease. Immunofluorescence staining with affinity-purified polyclonal rabbit antibody directed against human eosinophil granule major basic protein revealed marked tissue eosinophilia and abundant extracellular deposition of major basic protein in all specimens from 16 patients with IFT. By contrast, only occasional eosinophils and no extracellular major basic protein were detected in control thyroid tissues obtained from patients with multinodular goiter, Graves' disease, Hashimoto's disease, and normal thyroid tissue. The presence of marked eosinophil infiltration and extracellular major basic protein deposition in IFT and other associated fibrosclerotic conditions suggests a role for eosinophils and their products in propagating the fibrogenesis seen in IFT.

Potassium uptake, possibly together with chloride, is one of the presumed functions of Schwann cells in the peripheral nervous system. However, the presence of chloride channels has not been demonstrated in adult Schwann cells. We present here a new method which allows single channel recordings to be made from Schwann cells in situ without enzymatic treatment. Isolated rat spinal roots were split mechanically into several bundles. Within about 30 min after this procedure small belb-like vesicles (20-30 m in diameter) with a clean surface appeared at the edges of the fibre bundles. Immunofluorescence microscopy with a surface marker for Schwann cell membranes (monoclonal antibody O4) revealed that the vesicles originate from Schwann cells. In standard patch clamp recordings with symmetrical bath and pipette solutions (excised inside-out configuration) an anion channel with the following characteristics was mainly observed: (1) single channel slope conductance of 337 ± 5 pS in 125 mM KCl and 209 ± 6 pS in 125 mM K+ methylsulphate; (2) ion permeability ratio: PCl/PK/Pgluconate = 1/0.12/0.06; (3) linear current-voltage relationship (range ± 60 mV) and (4) voltage- and time-dependent inactivation (the channel was most active at potentials ± 20mV). Pharmacologically, the channel was completely blocked with zinc (1 mM) and barium (10 mM). A similar anion channel, showing characteristics 1 - (4), has been described in cultured Schwann cells of newborn rats (Gray et al., 1984). We now demonstrate that this channel is also present in adult Schwann cells in situ.

Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNAfor the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping CDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in control, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.

Sat, 1 Jan 1983 12:00:00 +0100 https://epub.ub.uni-muenchen.de/9316/1/9316.pdf Cremer, Thomas; Cremer, Marion; Grund, Christoph; Moll, R.; Franke, W. W.;

Antigens and corresponding sera were collected from travellers with leishmaniasis returning to Germany from different endemic areas of the old world. The antigenicity of these Leishmania strains, which were maintained in Syrian hamsters, was compared by indirect immunofluorescence (IFAT). Antigenicity was demonstrated by antibody titres in 18 sera from 11 patients. The amastigotic stages of nine strains of Leishmania donovani and four strains of Leishmania tropica were compared with each other and with the culture forms of insect flagellates (Strigomonas oncopelti and Leptomonas ctenocephali). Eighteen sera from 11 patients were available for antibody determination with these antigens. The maximal antibody titres in a single serum varied considerably depending on which antigen was used for the test. High antibody levels could only be maintained when Leishmania donovani was employed as the antigen, but considerable differences also occurred between the different strains of this species. The other antigens were weaker. No differences in antigenicity between amastigotes and promastigotes of the same strain were observed. It is important to select suitable antigens. Low titres may be of doubtful specificity and are a poor baseline for the fall in titre which is an essential index of effective treatment.