Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

Clinician treatment choices for post-traumatic stress disorder: ambassadors survey of psychiatrists in 39 European countries.

BACKGROUND: Considering the recently growing number of potentially traumatic events in Europe, the European Psychiatric Association undertook a study to investigate clinicians' treatment choices for post-traumatic stress disorder (PTSD). METHODS: The case-based analysis included 611 participants, who correctly classified the vignette as a case of PTSD, from Central/ Eastern Europe (CEE) (n = 279), Southern Europe (SE) (n = 92), Northern Europe (NE) (n = 92), and Western Europe (WE) (N = 148). RESULTS: About 82% woulduse antidepressants (sertraline being the most preferred one). Benzodiazepines and antipsychotics were significantly more frequently recommended by participants from CEE (33 and 4%, respectively), compared to participants from NE (11 and 0%) and SE (9% and 3%). About 52% of clinicians recommended trauma-focused cognitive behavior therapy and 35% psychoeducation, irrespective of their origin. In the latent class analysis, we identified four distinct "profiles" of clinicians. In Class 1 (N = 367), psychiatrists would less often recommend any antidepressants. In Class 2 (N = 51), clinicians would recommend trazodone and prolonged exposure therapy. In Class 3 (N = 65), they propose mirtazapine and eye movement desensitization reprocessing therapy. In Class 4 (N = 128), clinicians propose different types of medications and cognitive processing therapy. About 50.1% of participants in each region stated they do not adhere to recognized treatment guidelines. CONCLUSIONS: Clinicians' decisions for PTSD are broadly similar among European psychiatrists, but regional differences suggest the need for more dialogue and education to harmonize practice across Europe and promote the use of guidelines.

Natural languages and RNA virus evolution.

Information concepts from physics, mathematics and computer science support many areas of research in biology. Their focus is on objective information, which provides correlations and patterns related to objects, processes, marks and signals. In these approaches only the quantitative aspects of the meaning of the information is relevant. In other areas of biology, 'meaningful information', which is subjective in nature, relies on the physiology of the organism's sensory organs and on the interpretation of the perceived signals, which is then translated into action, even if this is only mental (in brained animals). Information is involved, in terms of both amount and quality. Here we contextualize and review the main theories that deal with 'meaningful-information' at a molecular level from different areas of natural language research, namely biosemiotics, code-biology, biocommunication and biohermeneutics. As this information mediates between the organism and its environment, we emphasize how such theories compare with the neo-Darwinian treatment of genetic information, and how they project onto the rapid evolution of RNA viruses.

Viruses as archaeological tools for uncovering ancient molecular relationships.

The entry of a virus into the host cell always implies the alteration of certain intracellular molecular relationships, some of which may involve the recovery of ancient cellular activities. In this sense, viruses are archaeological tools for identifying unexpressed activities in noninfected cells. Among these, activities that hinder virus propagation may represent cellular defense mechanisms, for example, activities that mutagenize the viral genome such as ADAR-1 or APOBEC activities. Instead, those that facilitate virus propagation can be interpreted as the result of viral adaptation to-or mimicking-cellular structures, enabling the virus to perform anthropomorphic activities, including hijacking, manipulating, and reorganizing cellular factors for their own benefit. The alternative we consider here is that some of these second set of cellular activities were already in the uninfected cell but silenced, under the negative control of the cell or lineage, and that they represent a necessary precondition for viral infection. For example, specifically loading an amino acid at the 3'-end of the mRNA of some plant viruses by aminoacyl-tRNA synthetases has proved essential for virus infection despite this reaction not occurring with cellular mRNAs. Other activities of this type are discussed here, together with the biological context in which they acquire a coherent meaning, that is, genetic latency and molecular conflict.

Archaeological approaches to RNA virus evolution.

Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures.

Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus.

The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC50) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Resultados de la técnica septum bisagra en desviación de la punta nasal

Introducción: La desviación de la punta nasal suele producir alteración estética y funcional nasal. Generalmente, asocia alteraciones morfológicas de los cartílagos alares, además de dismorfia del cartílago septal. Objetivo: Presentar una técnica quirúrgica alternativa para el tratamiento de la laterorrinia en tercio inferior nasal y sus resultados. Material y Método: Se trataron 27 pacientes con una laterorrinia en punta nasal con la técnica del septum bisagra, incluyendo la colocación de uno o dos injertos de expansión. Describimos el protocolo de anamnesis y exploración que seguimos en estos pacientes y la descripción detallada de la técnica quirúrgica. Resultados: En 19 pacientes se utilizó un injerto de expansión unilateral para completar la técnica y en ocho de forma bilateral. Se obtuvo una puntuación media en la escala visual analógica (EVA) de ventilación de 8,3, con una mejoría de seis puntos, y en la EVA de aspecto estético de 8,1, mejorando en 4,6 puntos. Conclusión: la utilización de la técnica del septum bisagra es beneficiosa para el tratamiento de la desviación de la punta nasal, tanto a nivel funcional como estético. Presenta ciertas diferencias frente a otras técnicas descritas. La indicación se debe adecuar a cada paciente de manera individualizada.

Introduction: Deviation of nasal tip usually produces aesthetic disturbance and nasal ventilation decrease. It is usually associated with alar cartilages morphologic disturbances and dysmorphia of the septal cartilage. Aim: To present an alternative surgical technique for treating laterorrhinia in the nasal inferior third and its outcomes. Material and Method: 27 patients with nasal tip laterorrhinia were treated with the hinge septum technique, including the placement of one or two spreader grafts. We describe the anamnesis and exploration protocol that we carried out in these patients and a detailed description of the surgical procedure. Results: Unilateral spreader graft was used in 19 patients to complete the technique, and bilateral spreader graft was used in eight. The average score in visual analogue scale (VAS) related to nasal flow was 8.3, which means an improvement of six points, and in VAS related to aesthetic appearance the score was 8.1, improving 4.6 points. Conclusion: The use of the hinge septum technique is useful to treat the deviation of the nasal tip in a functional and aesthetic way. There are several differences compared to other described techniques. The indication has to be suitable for each patient individually.

Application of archaeological concepts to the interpretation of RNA virus quasi species evolution.

An enduring problem concerning the evolution of RNA viruses stems from the fact that their long-term rates of evolution (substitutions/ site/year) are lower than those calculated by comparing sequences of isolates collected over short time periods or within a single host (shortterm or intra-host evolution). This inconsistency has been attributed to several reasons, including deviations from the assumption of a molecularclock (constancy of mutational inputs as a function of time) and variations in viral multiplication rates, among others. We previously proposed a non-phylogenetic method for extracting information contained in mRNAs, that cannot be identified from examination of primary sequences alone, and that we called «archaeological¼ information. In this new approach, mRNAs are of interest as molecules, not for their primary sequence or encoded proteins but for encrypted information established in a remote past. In the present article, we propose that an archaeological approach may also contribute to explain higher short-term than long-term evolution rates in RNA viruses, in this case, by using the archaeological concept of palimpsest. The palimpsest is a record of historical changes, but it is not a successively ordered or a complete record, rather it is the product of two opposing activities, one of writing and rewriting and the other of erasing. In RNA virus quasispecies, the gain or loss of mutations is reflected in changes in the submolar frequency of myriads of variants in the population. The fact that mutation elimination is not always complete, turns viral quasispecies into complex palimpsests of viral variants or sub-populations thereof. Here we relate two main different temporalities of the quasispecies palimpsest (short- and long-term) to the stability of mutations in response to changes related to three components of the virus: the virions, the infected cell and the host cell lineage. Host cell lineage-related viral memory would be mostly irre versible as they are adaptive products to host cell changes. In contrast, memories related to the environment of the virion or responsive to the environment of the infected cell, which is shortterm mutational input, is less constrained provided the alteration in the ancestral information carried by the RNA is only transient. The two intermixed memory components result in two differently contributing mutation rates whose influence in the final result depends on whether the timescales used to take the sequences for comparison are short or long term.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

Power output and hearing performance in osseointegrated auditory devices.

BACKGROUND AND OBJECTIVE: Osseointegrated auditory devices are hearing gadgets that use the bone conduction of sound to produce hearing improvement. The mechanisms and factors that contribute to this sound transmission have been widely studied, however, there are other aspects that remain unknown, for instance, the influence of the processor power output. The aim of this study was to know if there is any relationship between the power output created by the devices and the hearing improvement that they achieve. MATERIALS Y METHODS: 44 patients were implanted with a percutaneous Baha® 5 model. Hearing thresholds in pure tone audiometry, free-field audiometry, and speech recognition (in quiet and in noise) were measured pre and postoperatively in each patient. The direct bone conduction thresholds and the power output values from the processors were also obtained. RESULTS: The pure tone average threshold in free field was 39.29 dB (SD = 9.15), so that the mean gain was 29.18 dB (SD = 10.13) with the device. This involved an air-bone gap closure in 63.64% of patients. The pure tone average threshold in direct bone conduction was 27.6 dB (SD = 10.91), which was 8.4 dB better than the pure tone average threshold via bone conduction. The mean gain in speech recognition was 39.15% (SD = 23.98) at 40 dB and 36.66% (SD = 26.76) at 60 dB. The mean gain in the signal-to-noise ratio was -5.9 dB (SD = 4.32). On the other hand, the mean power output values were 27.95 dB µN (SD = 6.51) in G40 and 26.22 dB µN (SD = 6.49) in G60. When analysing the relationship between bone conduction thresholds and G40 and G60 values, a correlation from the frequency of 1000 Hz was observed. However, no statistically significant association between power output, functional gain or speech recognition gain was found. CONCLUSIONS: The osseointegrated auditory devices generate hearing improvement in tonal thresholds and speech recognition, even in noise. Most patients closed the air-bone gap with the device. There is a direct relationship between the bone conduction threshold and the power output values from the processor, but only in mid and high frequencies. However, the relationship between power output and gain in speech recognition is weaker. Further investigation of contributing factors is necessary.

Estudio del efecto de una formación basada en observación directa en la capacidad de mentalización del personal de guarderías / Estudi de l'efecte d'una formació basada en observació directa en la capacitat de mentalització del personal d'escoles bressol / Studying the effect of a training based on the direct observation on the mentalization capacity of kindergarten staff

Se ha demostrado que la alta capacidad de mentalización del personal educativo pre-escolar(PEd) incide en el desarrollo socioemocional de la teoría de la mente y del lenguaje del infante y que tiene efecto compensador en contextos sociales y familiares desfavorables. Pocos estudios evalúan los efectos de formaciones en la mentalización en PEd. Este estudio evaluó el efecto de una formación que promueve la observación y comprensión de la subjetividad propia y del infante en una muestra de 17 educadoras de tres guarderías de Barcelona. Se encuentra una mejora estadísticamente significativa en la mentalización global y diferentes dimensiones. Se proponen líneas de investigación que incluyan la mentalización en PEd y su impacto en la infancia.(AU)

The high mentalization capacity of pre-school education (PEd) staff has been shown to have an impact onthe socio-emotional development of the infant's theory of mind and language and to have a compensatory effect in unfavourable social and family contexts. Few studies have evaluated the effects of training on mentalization in ECD. This study evaluated the effect of training that promotes observation and understanding of self and infant subjectivity in a sample of 17 educators from three nurseries in Barcelona. A statistically significant improvement was found in global mentalization and different dimensions. Lines of research that include mentalization in PEdand its impact on children are proposed.(AU)

S'ha demostrat que l'alta capacitat de mentalització del personal educatiu preescolar (PEd)incideix en el desenvolupament socioemocional de la teoria de la ment i del llenguatge de l'infant i que té un efectecompensador en contextos socials i familiars desfavorables. Pocs estudis avaluen els efectes de formacions en lamentalització a PEd. Aquest estudi va avaluar l'efecte d'una formació que promou l'observació i la comprensióde la subjectivitat pròpia i de l'infant en una mostra de 17 educadores de tres escoles bressol de Barcelona. Hi hauna millora estadísticament significativa en la mentalització global i diferents dimensions. Es proposen línies derecerca que incloguin la mentalització a PEd i el seu impacte a la infància.(AU)

Análisis de la potencia de salida y del rendimiento auditivo de los dispositivos auditivos osteointegrados / Power output and hearing performance in osseointegrated auditory devices

Antecedentes y objetivos: Los dispositivos auditivos osteointegrados utilizan la transmisión del sonido por vía ósea para producir una mejoría auditiva. Los mecanismos y factores que intervienen en esta transmisión han sido ampliamente estudiados, sin embargo, existen otros aspectos que no conocemos, por ejemplo, la influencia que tiene la potencia de salida del procesador. El objetivo principal de este estudio fue conocer si existe alguna relación entre la potencia que emiten estos dispositivos y la mejoría auditiva que producen. Materiales y métodos: Hemos realizado un estudio en 44 pacientes portadores de un Baha® 5 percutáneo. De cada paciente se obtuvieron los umbrales de vía aérea y ósea en audiometría tonal liminar, en audiometría en campo libre, y en audiometría verbal en silencio y con ruido de fondo, tanto previa como posteriormente a la implantación. También se recogieron los umbrales de conducción ósea directa a través del procesador y los valores de ganancia en la potencia de salida del procesador. (AU)

Background and objective: Osseointegrated auditory devices are hearing gadgets that use the bone conduction of sound to produce hearing improvement. The mechanisms and factors that contribute to this sound transmission have been widely studied, however, there are other aspects that remain unknown, for instance, the influence of the processor power output. The aim of this study was to know if there is any relationship between the power output created by the devices and the hearing improvement that they achieve. Materials and methods: Forty-four patients were implanted with a percutaneous Baha® 5 model. Hearing thresholds in pure tone audiometry, free-field audiometry, and speech recognition (in quiet and in noise) were measured pre and postoperatively in each patient. The direct bone conduction thresholds and the power output values from the processors were also obtained. (AU)

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

Power output and hearing performance in osseointegrated auditory devices. / Análisis de la potencia de salida y del rendimiento auditivo de los dispositivos auditivos osteointegrados.

BACKGROUND AND OBJECTIVE: Osseointegrated auditory devices are hearing gadgets that use the bone conduction of sound to produce hearing improvement. The mechanisms and factors that contribute to this sound transmission have been widely studied, however, there are other aspects that remain unknown, for instance, the influence of the processor power output. The aim of this study was to know if there is any relationship between the power output created by the devices and the hearing improvement that they achieve. MATERIALS AND METHODS: Forty-four patients were implanted with a percutaneous Baha® 5 model. Hearing thresholds in pure tone audiometry, free-field audiometry, and speech recognition (in quiet and in noise) were measured pre and postoperatively in each patient .The direct bone conduction thresholds and the power output values from the processors were also obtained. RESULTS: The pure tone average threshold in free field was 39.29dB (SD 9.15), so that the mean gain was 29.18dB (SD 10.13) with the device. This involved an air-bone gap closure in 63.64% of patients. The pure tone average threshold in direct bone conduction was 27.6dB (SD 10.91), which was 8.4dB better than the pure tone average threshold via bone conduction. The mean gain in speech recognition was 39.15% (SD 23.98) at 40dB and 36.66% (SD 26.76) at 60dB. The mean gain in the signal-to-noise ratio was -5.9dB (SD 4.32). On the other hand, the mean power output values were 27.95dB µN (SD 6.51) in G40 and 26.22dB µN (SD 6.49) in G60. When analysing the relationship between bone conduction thresholds and G40 and G60 values, a correlation from the frequency of 1,000Hz was observed. However, no statistically significant association between power output, functional gain or speech recognition gain was found. CONCLUSIONS: The osseointegrated auditory devices generate hearing improvement in tonal thresholds and speech recognition, even in noise. Most patients closed the air-bone gap with the device. There is a direct relationship between the bone conduction threshold and the power output values from the processor, but only in mid and high frequencies. However, the relationship between power output and gain in speech recognition is weaker. Further investigation of contributing factors is necessary.