Molecular mapping of virus-infected cells with immunogold and metal-tagging transmission electron microscopy

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.This work has been funded by grant RTI2018-094445-B100 (MCIU/AEI/FEDER, UE) from the Ministry of Science and Innovation of Spain (C.R.). We are grateful to Ms. Sara Y. Fernández-Sánchez for critically reading the manuscript. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).S

Three-Dimensional Imaging of the Intracellular Assembly of a Functional Viral RNA Replicase Complex

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus–host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds

Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation

Modelos de valoración basados en magnitudes contables: limitaciones del modelo de resultado residual

Capital markets accounting research has recently redirected its interest towards valuation models. During the past decade, the Residual Income Valuation Model (RIVM) has become prominent in the literature. The objective of this paper is to analyze the conceptual foundations of the RIMV. After indicating its advantages in comparison with the traditional valuation models, we focus on the description of its limitations from a theoretical and empirical point of view. The most significant shortcomings which are analyzed are: the violation of the clean surplus relationship, the scale effect, and the estimation of terminal value

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation

Actas de las VI Jornadas Nacionales (JNIC2021 LIVE)

Estas jornadas se han convertido en un foro de encuentro de los actores más relevantes en el ámbito de la ciberseguridad en España. En ellas, no sólo se presentan algunos de los trabajos científicos punteros en las diversas áreas de ciberseguridad, sino que se presta especial atención a la formación e innovación educativa en materia de ciberseguridad, y también a la conexión con la industria, a través de propuestas de transferencia de tecnología. Tanto es así que, este año se presentan en el Programa de Transferencia algunas modificaciones sobre su funcionamiento y desarrollo que han sido diseñadas con la intención de mejorarlo y hacerlo más valioso para toda la comunidad investigadora en ciberseguridad

Paravertebral Muscle Mechanical Properties and Spinal Range of Motion in Patients with Acute Neck or Low Back Pain: A Case-Control Study

Our aims were to identify potential differences in muscle mechanical properties (MMPs) of cervical and lumbar tissues and in spinal range of motion (ROM) between patients with acute low back pain (LBP) or acute neck pain (NP) and healthy controls, and to identify if ROMs and MMPs are able to identify subjects among the three groups. Clinical variables (pain, disability, fear of movement, kinesiophobia, quality of life), MMPs and ROMs were obtained in 33 subjects with acute LBP, 33 with acute NP, and 33 healthy control subjects. Between-groups differences and explanatory models to discriminate groups depending on MMPs and ROMs were calculated. The results showed that cervical tone was higher in patients with acute NP than in controls, while cervical decrement was higher in both spinal pain groups. Patients with acute NP showed reduced cervical flexion when compared to acute LBP and control groups, and also cervical rotation, but just against controls. Furthermore, lumbar flexion was reduced in patients with acute LBP when compared to those with acute NP. Cervical decrement was able to discriminate spinal pain individuals from controls in a multinominal regression (R2: Cox–Snell estimation = 0.533; Nagelkerke estimation = 0.600). Lumbar flexion differentiated patients with acute LBP and controls, whereas cervical flexion differentiated patients with acute NP and controls. This study supports a tendency of the affectation of other spinal regions when only one is affected

Influence of Spinal Movements Associated with Physical Evaluation on Muscle Mechanical Properties of the Lumbar Paraspinal in Subjects with Acute Low Back Pain

This research aimed to identify changes in muscle mechanical properties (MMPs) when a standardized sequence of movements is performed and to determine the influence of acute low back pain (LBP) and age on the MMPs. Socio-demographic, clinical variables and MMPs were collected in 33 patients with LBP and 33 healthy controls. A 2 × 2 × 2 (group × age × time) analysis of variance (ANOVA) mixed model was used to determine the effect of the study factors on the different MMPs. There were no significant triple interactions. After the movements, tone and stiffness increased 0.37 Hz and 22.75 N/m, respectively, in subjects <35 years, independent of their clinical status. Relaxation showed differences by age in healthy subjects and creep in LBP subjects. Furthermore, elasticity was higher in <35 years (p < 0.001) without the influence of any other factor. In conclusion, sequenced movements can modify tone and stiffness as a function of age, while age-associated changes in viscoelastic characteristics depends on pain but not on movements. The MMPs should be assessed, not only at the beginning of the physical examination at rest, but also along the patient’s follow-up, depending on their pain and age, in a clinical setting

Teaching empathy to nursing students: A randomised controlled trial

Background: Empathy has been reported to produce a positive effect on improving patient health outcomes, becoming a fundamental skill in any health personnel-patient relationship. Objective: To evaluate the effectiveness of an intervention designed to improve the nursing students' empathy, the learning perception, the improvement of the perception in the understanding of the content, and in the degree of difficulty as well as the acquisition of skills. Design: Multicentre randomised controlled trial. Setting: This research was conducted at two schools of nursing at a public university in the Southwest of Spain. Participants: 116 nursing students were randomly assigned to an experimental ora control group (delayed intervention group once the post-training analysis was completed) during the second semester of the 2015/ 2016 academic year. Methods: Pre-test, post-test, and follow-up data were obtained for each group using a simulated clinical interview. Empathy was the primary outcome (The Consultation and Relational Empathy Measure, Jefferson Scale of Empathy student version, Reynolds Empathy Scale, and Carkhuff Scale). The students' perceived knowledge, the learning perception, the self-esteem (Rosenberg Self-Esteem Scale) and the understanding of the content and acquisition of skills (ad-hoc questions) were also analysed. Results: The results were improved in ali the measures conducted in the experimental groups at the different centres after the intervention. The mean post-test simulation seores were higher than the pre-test with statistically significan! differences. The results were maintained in the follow-up. The student's perception of learning and the perception of understanding of the content and the acquisition of ski lls were improved as well. Conclusion: The study support that training in empathic competence is effective

Evidence of the use of threshing-boards in the spanish Meseta during the Copper Age. Microwear analysis of fl int denticulates from ‘the ditched enclosure’ of El Casetón de la Era (Villalba de los Alcores, Valladolid)

La presencia de grandes piezas líticas talladas bifacialmente es una constante en los contextos de la Edad del Cobre en el interior de la Península Ibérica. El brillo apreciable a simple vista en su filo denticulado ha sido atribuido a su uso como hoz. El estudio que ahora presentamos contradice parcialmente esta propuesta, ya que la mayor parte de las piezas halladas en el yacimiento de El Casetón de la Era (Villalba de los Alcores, Valladolid) no eran hoces sino elementos de trillo. Estamos ante las evidencias más antiguas de su uso en la Península Ibérica. Este tipo de piezas tienen cierta semejanza con las encontradas en el Próximo Oriente durante la Edad del BronceLarge bifacial fl int tools are frequently found in Copper Age sites from Iberian Península. They usually show a visible gloss on their denticulated working edges, possibly related to cereal cutting. As a result, these tools have been interpreted as sickle blades. This paper partially contradicts this hypothesis, since most of the pieces recovered at the site of El Casetón de la Era (Villalba de los Alcores, Valladolid) were not used as sickles, but rather they could have been fl akes in threshing-boards. Therefore, this is the earliest evidence of the use of threshingboards in Iberia. This type of tool is in some way similar to those found in the Near East during the Bronze Age