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The aim of this study was to evaluate and compare the biofilm formation properties of common pathogens associated with implant-related infections on two different implant material types. Bacterial strains tested in this study were Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli. Implant materials tested and compared were PLA Resorb × polymer of Poly DL-lactide (PDLLA) comprising 50% poly-L-lactic acid and 50% poly-D-lactic acid) and Ti grade 2 (tooled with a Planmeca CAD-CAM milling device). Biofilm assays were done with and without saliva treatment to evaluate the effect of saliva on bacterial adhesion and to mimic the intraoral and extraoral surgical routes of implant placement, respectively. Five specimens of each implant type were tested for each bacterial strain. Autoclaved material specimens were first treated with 1:1 saliva-PBS solution for 30 min, followed by washing of specimens and the addition of bacterial suspension. Specimens with bacterial suspension were incubated for 24 h at 37 °C for biofilm formation. After 24 h, non-adhered bacteria were removed, and specimens were washed, followed by removal and calculation of adhered bacterial biofilm. S. aureus and E. faecalis showed more attachment to Ti grade 2, whereas S. mutans showed higher adherence to PLA in a statistically significant manner. The salivary coating of specimens enhanced the bacterial attachment by all the bacterial strains tested. In conclusion, both implant materials showed significant levels of bacterial adhesion, but saliva treatment played a vital role in bacterial attachment, therefore, saliva contamination of the implant materials should be minimized and considered when placing implant materials inside the body. [ABSTRACT FROM AUTHOR]

Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs. [ABSTRACT FROM AUTHOR]

This study investigated two of the most commonly used CAD–CAM materials for patient-specific reconstruction in craniomaxillofacial surgery. The aim of this study was to access the biofilm formation of Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli on titanium and PEEK medical implant materials. Two titanium specimens (titanium grade 2 tooled with a Planmeca CAD–CAM milling device and titanium grade 5 tooled with a computer-aided design direct metal laser sintering device (CAD-DMLS)) and one PEEK specimen tooled with a Planmeca CAD–CAM milling device were studied. Bacterial adhesion on implants was evaluated in two groups (saliva-treated group and non-saliva-treated group) to imitate intraoral and extraoral surgical routes for implant placement. The PEEK medical implant material showed higher bacterial adhesion by S. aureus, S. mutans, and E. coli than titanium grade 2 and titanium grade 5, whereas E. faecalis showed higher adhesion to titanium as compared to PEEK. Saliva contamination of implants also effected bacterial attachment. Salivary coating enhanced biofilm formation by S. aureus, S. mutans, and E. faecalis. In conclusion, our findings imply that regardless of the implant material type or tooling techniques used, salivary coating plays a vital role in bacterial adhesion. In addition, the majority of the bacterial strains showed higher adhesion to PEEK than titanium. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the biofilm formation of Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli on titanium implants with CAD-CAM tooling techniques. Twenty specimens of titanium were studied: Titanium grade 2 tooled with a Planmeca CAD-CAM milling device (TiGrade 2), Ti6Al4V grade 5 as it comes from CAD-DMLS device (computer aided design-direct metal laser sintering device) (TiGrade 5), Ti6Al4V grade 23 as it comes from a CAD-CAM milling device (TiGrade 23), and CAD-DMLS TiGrade 5 polished with an abrasive disc (TiGrade 5 polished). Bacterial adhesion on the implants was completed with and without saliva treatment to mimic both extraoral and intraoral surgical methods of implant placement. Five specimens/implant types were used in the bacterial adhesion experiments. Autoclaved implant specimens were placed in petri plates and immersed in saliva solution for 30 min at room temperature and then washed 3× with 1× PBS. Bacterial suspensions of each strain were made and added to the specimens after saliva treatment. Biofilm was allowed to form for 24 h at 37 °C and the adhered bacteria was calculated. Tooling techniques had an insignificant effect on the bacterial adhesion by all the bacterial strains studied. However, there was a significant difference in biofilm formation between the saliva-treated and non-saliva-treated implants. Saliva contamination enhanced S. mutans, S. aureus, and E. faecalis adhesion in all material types studied. S. aureus was found to be the most adherent strain in the saliva-treated group, whereas E. coli was the most adherent strain in the non-saliva-treated group. In conclusion, CAD-CAM tooling techniques have little effect on bacterial adhesion. Saliva coating enhances the biofilm formation; therefore, saliva contamination of the implant must be minimized during implant placement. Further extensive studies are needed to evaluate the effects of surface treatments of the titanium implant on soft tissue response and to prevent the factors causing implant infection and failure. [ABSTRACT FROM AUTHOR]

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis. [ABSTRACT FROM AUTHOR]

We examined the distribution of selected raft proteins on the sarcolemma of skeletal myofibers and the role of cholesterol environment in the distribution. Immunofluorescence staining showed that flotillin-1 and influenza hemagglutinin exhibited rafts that located in the domains deficient of the dystrophin glycoprotein complex, but the distribution patterns of the two proteins were different. Cholesterol depletion from the sarcolemma by means of methyl-β-cyclodextrin resulted in distorted caveolar morphology and redistribution of the caveolin 3 protein. Concomitantly, the water permeability of the sarcolemma increased significantly. However, cholesterol depletion did not reshuffle flotillin 1 or hemagglutinin. Furthermore, a hemagglutinin variant that lacked a raft-targeting signals exhibited a similar distribution pattern as the native raft protein. These findings indicate that each raft protein exhibits a strictly defined distribution in the sarcolemma. Only the distribution of caveolin 3 that binds cholesterol was exclusively dependent on cholesterol environment. [ABSTRACT FROM AUTHOR]

A comparative application of major dynamic light scattering (DLS)‐based image methodologies applied to transcranial cerebral blood flow imaging is presented. In particular, the study delves into assessing capability of Laser Doppler Flowmetry (LDF), Laser Speckle Contrast Imaging (LSCI), and Diffuse Correlation Spectroscopy (DCS) in enhancing the spatial and temporal resolution of transcranial blood flow imaging. An integral part of the study is focused on the modulation of blood flow through the administration of the vasodilator drug, Sodium Nitroprusside (SNP). This pharmacological intervention facilitated a direct observation of cerebral vasculature's responsiveness to external stimuli, illuminating the physiological adaptations within the brain's microvascular architecture. Advanced LSCI processing techniques are incorporated, notably entropy and principal component analysis (PCA). Entropy is providing a quantifiable measure of the randomness and complexity within the speckle patterns of transcranial blood flow images, revealing remarkably similar outcomes with DSC approach in terms of blood flow dynamics and its quantitative evaluation. The application of PCA approach is provided a more nuanced understanding of blood flow dynamics, facilitating the identification of subtle changes induced by drug administration. This method proved instrumental in enhancing the visualization and detection of nuanced blood flow dynamics, thereby allowing for a more detailed examination of cerebral circulation alterations induced by SNP administration. The study seeks to offer a wider‐ranging insight into comprehending the translating further the concept of DLS into transcrainial blood flow vizualization and explore its practical applications, considering hardware, advanced quantitative image processing, and data acquisition. [ABSTRACT FROM AUTHOR]

Epithelial cell adhesion is mediated by actin cytoskeleton-linked focal adhesions (FAs) and intermediate filament-associated hemidesmosomes (HDs). HDs are formed by α6β4-integrins and mediate stable anchoring to the extracellular matrix (ECM) while FAs containing β1-integrins regulate cell migration. Loss of HDs has been reported in various cancers such as prostate cancer where it correlates with increased invasive migration. Here we have studied cell migration properties and FA dynamics in genetically engineered prostate epithelial cell lines with intact or disrupted HDs. Disruption of HDs by depleting α6- or β4-integrin expression promoted collective cell migration and modulated migratory activity. Dynamic analysis of fluorescent protein-tagged FA marker proteins revealed faster FA assembly and disassembly kinetics in HD-depleted cells. FRAP analysis showed that loss of HDs correlated with faster diffusion rates of focal adhesion kinase (FAK) and vinculin in and out of FAs. These data suggest that loss of α6β4-mediated HDs promote cell migration and FA assembly dynamics by influencing the molecular diffusion rates of FAK., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schmidt, Kaakinen, Wenta and Manninen.)

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2 443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2 443 ), a genetic model for breast cancer and metastasis (MMTV- PyMT ), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2 443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2 443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2 443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro , ANGPT2 443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2 DAP ) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer. See related commentary by Kamiyama and Augustin, p. 35 ., (©2020 American Association for Cancer Research.)

The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found Angpt4 expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of Angpt4 resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function., Competing Interests: HE, MK, EK, MK, IM, SR, MC, SM, MN, LE No competing interests declared, (© 2018, Elamaa et al.)

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.