Transmission electron microscopy (TEM) has been essential to study virus–cell interactions. The architecture of viral replication factories, the principles of virus assembly and the components of ...virus egress pathways are known thanks to the contribution of TEM methods. Specially, when studying viruses in cells, methodologies for labeling proteins and other macromolecules are important tools to correlate morphology with function. In this review, we present the most widely used labeling method for TEM, immunogold, together with a lesser known technique, metal‐tagging transmission electron microscopy (METTEM) and how they can contribute to study viral infections. Immunogold uses the power of antibodies and electron dense, colloidal gold particles while METTEM uses metallothionein (MT), a metal‐binding protein as a clonable tag. MT molecules build gold nano‐clusters inside cells when these are incubated with gold salts. We describe the necessary controls to confirm that signals are specific, the advantages and limitations of both methods, and show some examples of immunogold and METTEM of cells infected with viruses.
Main steps of molecular mapping using Immunogold (on the left) and METTEM (on the right) before TEM. The combination of both methods (center) provides a powerful tool to study virus–cell interactions. Created with BioRender.com.
Abstract Background Oral anticoagulation reduces the risk of mortality in atrial fibrillation (AF), but examination of the causes of death is essential to design new strategies to further reduce the ...high mortality rates observed in this population. Objectives The authors sought to analyze and compare causes of death in patients receiving direct oral anticoagulants (DOAC) or warfarin for prevention of stroke and systemic embolism (SE) in AF. Methods The authors systematically searched for randomized trials of DOAC versus warfarin for prevention of stroke/SE in AF. The main outcome was mortality and independently adjudicated specific causes of death. The authors used the random effects model of meta-analysis to combine the studies. Results 71,683 patients from 4 trials were included (134,046 patient-years of follow-up). A total of 6,206 patients (9%) died during follow-up. Adjusted mortality rate was 4.72%/year (95% confidence interval CI: 4.19 to 5.28). Cardiac deaths accounted for 46% of all deaths, whereas nonhemorrhagic stroke/SE and hemorrhage-related deaths represented 5.7% and 5.6% of the total mortality, respectively. Compared with patients who were alive, those who died had more frequent history of heart failure (odds ratio OR: 1.75; 95% CI: 1.25 to 2.44), permanent/persistent AF (OR: 1.38; 95% CI: 1.25 to 1.52) and diabetes (OR: 1.37; 95% CI: 1.11 to 1.68); were more frequently male (OR: 1.24; 95% CI: 1.13 to 1.37) and older (mean difference 3.2 years; 95% CI: 1.6 to 4.8); and had a lower creatinine clearance (−9.9 ml/min; 95% CI: −11.3 to −8.4). There was a small, but significant, reduction in all-cause mortality with the DOAC versus warfarin (difference −0.42%/year; 95% CI: −0.66 to −0.18), mainly driven by a reduction in fatal bleedings. Conclusions In contemporary AF trials, most deaths were cardiac-related, whereas stroke and bleeding represented only a small subset of deaths. Interventions beyond anticoagulation are needed to further reduce mortality in AF.
Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during ...surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.
Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important ...components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.
The aim of this paper is to derive an index able to indicate if a discount function exhibits increasing or decreasing impatience, and, even, in the last case, whether the decreasing impatience is ...moderate or strong. Moreover, it will be shown that the sign of this indicator coincides with the sign of the convexity index of the discount function when only considering the cases of increasing and decreasing impatience. Consequently, this parameter supposes an improvement of Prelec's index of convexity. The main advantage of this novel measure is that, the same as Prelec's index, it uses the differential calculus and, moreover, can be easily plotted by showing the changes from a type of impatience to another one according to time.
En la actualidad los estudios de joyería están cobrando cada vez más importancia. Las alhajas no solo servían para mostrar el poder que tenía su dueño, sino que contenían información acerca de los ...intereses y la personalidad de quien lo llevaba. Estas fueron utilizadas como un instrumento de prestigio por las élites. Desde fechas tempranas las mujeres comenzaron a usarlas para subrayar sus cualidades, apareciendo en estos pequeños objetos preciosos alusiones al carácter de su dueña. Uno de los motivos que comenzó a cobrar fuerza en las alhajas fue la representación de una virtud: la Prudencia. Aunque los tipos iconográficos de la Prudencia han variado con el paso del tiempo, su presencia en joyeles, pinjantes o insignias de sombrero aluden a la virtud de su portadora. Así, mediante las piezas que han llegado hasta la actualidad podremos analizar el papel que cumplieron.
Transport of neo-synthesized influenza A virus (IAV) viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the precise mechanism remains poorly understood. We ...used metal-tagging and immunolabeling to visualize viral proteins and cellular endomembrane markers by electron microscopy of IAV-infected cells. Unexpectedly, we provide evidence that the vRNP components and the Rab11 protein are present at the membrane of a modified, tubulated endoplasmic reticulum (ER) that extends all throughout the cell, and on irregularly coated vesicles (ICVs). Some ICVs are found very close to the ER and to the plasma membrane. ICV formation is observed only in infected cells and requires an active Rab11 GTPase. Against the currently accepted model in which vRNPs are carried onto Rab11-positive recycling endosomes across the cytoplasm, our findings reveal that the endomembrane organelle that is primarily involved in the transport of vRNPs is the ER.
Objective
To assess the clinical outcomes of flapless, immediate implant placement with simultaneous augmentation of dehiscence defects and immediate provisional restoration.
Materials and Methods
...Retrospective evaluation of 40 periodontal patients who received a flapless immediate implant for single‐tooth replacement with simultaneous augmentation of dehiscence defects and immediate provisional restoration, at follow‐up periods ranging from 1 year minimum to 7 years maximum. Dehiscence defects were augmented using autogenous bone grafts and/or deproteinized bovine bone mineral, and autogenous or allogeneic connective tissue grafts. The following aspects were evaluated: clinical success (Clinical Oral Implants Research, 8, 1997, 161), crestal bone loss (calibrated digital periapical X‐rays) and aesthetics (Pink Esthetic Score, PES). Additionally, a questionnaire was devised to obtain patient's subjective evaluation.
Results
A 98.3% (95% CI: 91%–99%) success rate was observed. At 8 weeks, mean bone loss was 0.47 mm (n = 39); between 8 weeks and 1 year, this va lue attained 0.24 mm and between years 1 and 5 years 0.06 mm (n = 9). The evaluated PES in 37 single‐tooth implants was 12.4, with PES = 7 (aesthetic failures) in two cases, and aesthetic outcomes were excellent (PES ≥ 12) in 29 cases (78.4%). When compared with the contralateral tooth, a recession under 1 mm was observed in 78% of cases. The patient's subjective satisfaction value ranged from 98% to 100%.
Conclusion
This type of minimally invasive treatment of alveoli with buccal plate deficiencies delivers, in the hands of an experienced clinician, a high clinical and aesthetic success percentage being rated by patients as satisfactory. Clinicians should be aware that 1–2 mm comparative recession could be observed.
The purpose of this study was to determine how preterm white matter injury (WMI) and long-term thalamic growth interact to predict 8-year neurodevelopmental outcomes.
A prospective cohort of 114 ...children born at 24 to 32 weeks' gestational age (GA) underwent structural and diffusion tensor magnetic resonance imaging early in life (median 32 weeks), at term-equivalent age and at 8 years. Manual segmentation of neonatal WMI was performed on T1-weighted images and thalamic volumes were obtained using the MAGeT brain segmentation pipeline. Cognitive, motor, and visual-motor outcomes were evaluated at 8 years of age. Multivariable regression was used to examine the relationship among neonatal WMI volume, school-age thalamic volume, and neurodevelopmental outcomes.
School-age thalamic volumes were predicted by neonatal thalamic growth rate, GA, sex, and neonatal WMI volume (p < 0.0001). After accounting for total cerebral volume, WMI volume remained associated with school-age thalamic volume (β = -0.31, p = 0.005). In thalamocortical tracts, fractional anisotropy (FA) at term-equivalent age interacted with early WMI volume to predict school-age thalamic volumes (all p < 0.02). School-age thalamic volumes and neonatal WMI interacted to predict full-scale IQ (p = 0.002) and adverse motor scores among those with significant WMI (p = 0.01). Visual-motor scores were predicted by thalamic volumes (p = 0.04).
In very preterm-born children, neonatal thalamic growth and WMI volume predict school-age thalamic volumes. The emergence at term of an interaction between FA and WMI to impact school-age thalamic volume indicates dysmaturation as a mechanism of thalamic growth failure. Cognition is predicted by the interaction of WMI and thalamic growth, highlighting the need to consider multiple dimensions of brain injury in these children. ANN NEUROL 2021;90:584-594.
PDF
