Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their coagulation and adhesion to the device surface. One possible way to achieve this is to use scanning electron microscopy (SEM). The aim was to develop a novel SEM-based method to assess the thrombogenic potential of medical devices and their adhesiveness to blood cells. As a part of this task, also find a convenient procedure of efficient and non-destructive sample fixation for SEM while reducing the use of highly toxic substances and shortening the fixation time. A polymeric surgical mesh was exposed to blood so that blood elements adhered to its surface. Such prepared samples were then chemically fixed for a subsequent SEM measurement; a number of fixation procedures were tested to find the optimal one. The fixation results were evaluated from SEM images, and the degree of blood elements' adhesion was determined from the images using ImageJ software. The best fixation was achieved with the May-Grünwald solution, which is less toxic than chemicals traditionally used. Moreover, manipulation with highly toxic osmium tetroxide can be avoided in the proposed procedure. A convenient methodology for SEM image analysis has been developed too, enabling to quantitatively evaluate the interaction of blood with the surfaces of various medical devices. Our method replaces the subjective assessment of surface coverage with a better-defined procedure, thus offering more precise and reliable results.
- MeSH
- histologické techniky * MeSH
- mikroskopie elektronová rastrovací MeSH
- oxid osmičelý * MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.
- MeSH
- adenovirové infekce lidí metabolismus virologie MeSH
- biologické modely MeSH
- elektronová kryomikroskopie MeSH
- internalizace viru * MeSH
- laktoferrin * chemie genetika metabolismus ultrastruktura MeSH
- lidé MeSH
- lidské adenoviry * chemie genetika metabolismus ultrastruktura MeSH
- mutace MeSH
- respirační sliznice cytologie metabolismus virologie MeSH
- rozpustnost MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- virové plášťové proteiny * chemie genetika metabolismus ultrastruktura MeSH
- virové receptory * chemie genetika metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. METHODS: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. RESULTS: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. CONCLUSIONS: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.
- MeSH
- elektronová kryomikroskopie MeSH
- exprese genu MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- fosforylace MeSH
- insulinu podobný růstový faktor I * genetika metabolismus MeSH
- inzulin metabolismus MeSH
- lidé MeSH
- myši MeSH
- protein - isoformy metabolismus MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- receptor IGF typ 1 * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.
- MeSH
- antibakteriální látky terapeutické užití MeSH
- aplikace inhalační MeSH
- cilie patologie MeSH
- dechová cvičení metody MeSH
- diferenciální diagnóza MeSH
- elektronová mikroskopie metody MeSH
- genetické testování metody MeSH
- infekce dýchací soustavy etiologie MeSH
- infertilita etiologie MeSH
- lidé MeSH
- oxid dusnatý analýza MeSH
- poruchy ciliární motility * diagnóza patologie terapie MeSH
- situs inversus etiologie MeSH
- videomikroskopie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Bacteriophages of Borrelia burgdorferi are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. No virulent borrelial viruses have been identified, but all B. burgdorferi isolates carry a prophage φBB1 as resident circular plasmids. Like its host, the φBB1 phage is quite distinctive and shares little sequence similarity with other known bacteriophages. We expressed φBB1 head morphogenesis proteins in Escherichia coli which resulted in assembly of homogeneous prolate procapsid structures and used cryo-electron microscopy to determine the three-dimensional structure of these particles. The φBB1 procapsids consist of 415 copies of the major capsid protein and an equal combined number of three homologous capsid decoration proteins that form trimeric knobs on the outside of the particle. One of the end vertices of the particle is occupied by a portal assembled from twelve copies of the portal protein. The φBB1 scaffolding protein is entirely α-helical and has an elongated shape with a small globular domain in the middle. Within the tubular section of the procapsid, the internal scaffold is built of stacked rings, each composed of 32 scaffolding protein molecules, which run in opposite directions from both caps with a heterogeneous part in the middle. Inside the portal-containing cap, the scaffold is organized asymmetrically with ten scaffolding protein molecules bound to the portal. The φBB1 procapsid structure provides better insight into the vast structural diversity of bacteriophages and presents clues of how elongated bacteriophage particles might be assembled.
- MeSH
- elektronová mikroskopie MeSH
- lidé MeSH
- ortodontické zámky škodlivé účinky MeSH
- premolár MeSH
- zubní nástroje MeSH
- zubní sklovina * ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinická studie MeSH
Avian (ortho)reovirus (ARV), which belongs to Reoviridae family, is a major domestic fowl pathogen and is the causative agent of viral tenosynovitis and chronic respiratory disease in chicken. ARV replicates within cytoplasmic inclusions, so-called viral factories, that form by phase separation and thus belong to a wider class of biological condensates. Here, we evaluate different optical imaging methods that have been developed or adapted to follow formation, fluidity and composition of viral factories and compare them with the complementary structural information obtained by well-established transmission electron microscopy and electron tomography. The molecular and cellular biology aspects for setting up and following virus infection in cells by imaging are described first. We then demonstrate that a wide-field version of fluorescence recovery after photobleaching is an effective tool to measure fluidity of mobile viral factories. A new technique, holotomographic phase microscopy, is then used for imaging of viral factory formation in live cells in three dimensions. Confocal Raman microscopy of infected cells provides "chemical" contrast for label-free segmentation of images and addresses important questions about biomolecular concentrations within viral factories and other biological condensates. Optical imaging is complemented by electron microscopy and tomography which supply higher resolution structural detail, including visualization of individual virions within the three-dimensional cellular context.
Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.
- MeSH
- elektronová mikroskopie MeSH
- Leishmania * MeSH
- Psychodidae * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Úvod: Dnešní trh nabízí uživatelům velké množství manuálních zubních kartáčků lákavých barev, odlišných designů hlaviček, rukojetí, zastřižení a měkkosti vláken, ale i rozličných cen. Je však drahý, ekologický či pestrobarevný kartáček kvalitní a nepoškozuje nás? Kvalitní kartáček je definován jako kartáček, který svými vlákny nepoškozuje dásně ani tvrdé zubní tkáně. Tato studie je proto zaměřena na kvalitu opracování konců vláken. Cílem je čtenáři ukázat a porovnat kvalitu vláken šesti odlišných výrobků prodávaných na našem trhu. Metodika: Pro tuto studii bylo vybráno šest kartáčků od šesti různých výrobců: A. Curaprox 5460; B. Dr.Max PRO32 Extra soft 5400; C. Tepe Compact x – soft; D. Spokar X 5500 Ultrasoft; E. SOFTdent ECO Ultrasoft; F. Elmex Ultrasoft. Kartáčky byly vybrány se stejnými parametry, jako je rovné zastřižení a měkkost vláken. Kvalita konců vláken kartáčků byla detekována pomocí stereomikroskopie a elektronové mikroskopie. Výsledky: Vybrané manuální kartáčky vykazují velkou diskrepanci kvality opracování vláken nejen při porovnávání mezi pozorovanými kartáčky, ale kvalita vláken není konzistentní ani v rámci jednoho pozorování jednoho kartáčku. Závěr: Pozorování jasně prokázalo výrazně vyšší kvalitu opracování vláken u kartáčku Tepe Compact x – soft v porovnání s ostatními kartáčky.
Introduction: Today's market offers to users a large number of manual toothbrushes in attractive colors, different head designs, handles, bristle cuts as well as softness, and various prices too. However, is an expensive, ecological, or brightly coloured toothbrush of a good quality and does it not harm us? A quality toothbrush is defined as a toothbrush whose bristles do not damage the gums or hard dental tissues. This study is focused on the quality of bristle ends processing. The aim is to show the reader and compare the bristle quality of six different products sold on our market. Methods: Six toothbrushes from six different manufacturers were selected for this study: A. Curaprox 5460; B. Dr.Max PRO32 Extra soft 5400; C. Tepe Compact x – soft; D. Spokar X 5500 Ultrasoft; E. SOFTdent ECO Ultrasoft; F. Elmex Ultrasoft. Brushes were selected with the same parameters such as straight cutting and softness of the bristles. The quality of brush bristle ends was detected using stereo microscopy and electron microscopy. Results: The selected manual brushes show a large discrepancy in the quality of the processing of the bristles not only in the comparison amongst the observed brushes, but the quality of the bristles is not consistent even within one observation of a single brush. Conclusion: The observation clearly demonstrated a significantly higher quality of the bristles processing of the Tepe Compact x – soft brush compared to other brushes.
- MeSH
- elektronová mikroskopie MeSH
- lidé MeSH
- mikroskopie MeSH
- zubní prostředky domácí * MeSH
- Check Tag
- lidé MeSH
